Megakaryocytic particles and microparticles for cell therapy and fate modification of stem and progenitor cells

ABSTRACT

Applications in transfusion medicine requiring platelets, and hematopoietic stem-cell transplantations require either platelets or enhancement of in vivo platelet biogenesis. Gene therapy applications of hematopoietic stem and progenitor cells (HSPCs) require effective and specific modification of HSPCs by DNA, RNA or other biological molecules. Here we disclose methods for the generation, and modification of megakaryocytic microparticles (MkMPs) or microvesicles, that can be used in the aforementioned transfusion and transplantation medicine applications and in gene therapy applications involving hematopoietic stem cells. The biological effects of modified or unmodified MkMPs have never been previously disclosed and thus, this invention claims all biological applications of MkMPs in in vivo therapeutic applications or ex vivo applications to produce various cells and cell parts, modify various target cells or deliver molecules including drugs to HSPCs and related cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/000,109, filed 19 May 2014, the contents of which are incorporatedherein by reference.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This work is supported by a grant from the National Institutes of Health(Award No. R21HL106397). The United States has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to the generation and use of megakaryocyticmicroparticles (MkMPs) or microvesicles, platelet-like particles (PLPs)and proplatelets/preplatelets (PPTs) that are produced from maturemegakaryocytes. Megakaryocytes can be produced from human hematopoieticstem and progenitor cells (HSPCs) but also from embryonic and inducedpluripotent stem (iPS) cells. The present invention relates to allapplications MkMPs in Transfusion Medicine, hematopoetic-celltransplantation and for delivering DNA, RNA, protein and other moleculesto HSPCs (hematopoietic stem/progenitor cells) that can be obtained fromvarious sources including the bone marrow, the peripheral blood, cordblood or from embryonic or iPS cells. The present invention also relatesto methods for using biomechanical forces as a mean to increase thenumber of PLPs, PPTs and MkMPs that can be generated frommegakaryocytes. The present invention also relates to the use of certainbioreactor setting for generating microparticles from various cell typesother than megakaryocytes.

Megakaryocytes (Mks) are derived from hematopoietic (blood) stem cells(typically contained in the CD34⁺ compartment), and are distinguished bytheir very large size, high DNA content, and the formation ofproplatelet extensions which shed platelets, the small cells necessaryfor blood coagulation. Mk cells differentiate in the bone-marrow (BM)vasculature: they undergo a variation of the normal cell cycle, termedendomitosis, to form polyploid cells (≥8N DNA content). Committed Mksmigrate from the hematopoietic compartment of BM towards the endotheliallining of the BM sinusoids where they mature and extend long, branchedcytoplasmic protrusions termed proplatelets through gaps of theendothelium into the vasculature. Mk cells encounter mechanical stressesas they deform to penetrate the gaps of the sinusoid walls, and shearforces by the exposure to blood flow. The pulmonary circulation isanother important site of Mk maturation and platelet biogenesis: Mks mayenter the BM circulation and reach the lungs where they shedproplatelets. As such, Mk cells encounter shear forces in circulation aswell as mechanical strain in the lung vasculature. Thus, Mk maturationand platelet release appear to be stress-induced processes. However, thecellular/molecular events underlying the effects of mechanical stresseson Mk maturation and platelet biogenesis remain unexplored from thefundamental and practical applications point of view.

Platelets are an expensive product in limited supply. This is due to thecollection and processing steps from donated blood, and the fact thatplatelets cannot be frozen, but also due to the possibility of bacterialor blood-borne pathogen contamination, and of alloimmunization ofrecipients. As was recently reviewed and argued, culture-derivedplatelets, produced under Good Manufacturing Practices, hold a greatpotential for providing an abundant, safer and more tolerated plateletsupply for transfusion therapies. However, major advances are needed forlarge-scale, culture-based platelet production to become economicallyattractive. This will require improvements in the expansion of CD34⁺cells into Mks, and the ability to produce large, polyploid Mk cells,since the number of platelets produced is proportional to the cellploidy. As Mk maturation is also affected by interactions with stromaand extracellular matrix, platelet production will need to engagebioreactor systems involving semi-synthetic matrices under controlledflow conditions to simulate, to the extent possible, in vivo conditions.

In addition to platelet transfusions, there is a need to enhanceplatelet biogenesis in patients with thrombotic deficiency or excessivebleeding due to trauma, also in patients undergoing chemotherapytreatment for cancer due to the fact that chemotherapy destroys theability of the body to produce platelets. In vitro production offunctional proplatelets/platelets is firmly established. Thus,culture-derived Mks or platelets could provide a safer and moretolerated supply for transfusion therapies greatly impacting a verylarge community of patients in the US and worldwide. Calculationssuggest that generation of clinically-relevant doses of functionalplatelets is possible, but key scientific and technological challengesremain. First, expansion of hematopoietic stem cells without loss ofMk-differentiation potential, and then production of a larger number ofMks per input CD34⁺ cell will require substantial improvements. Second,because the degree of ploidy directly correlates with the number ofplatelets produced, it is necessary to increase the ploidy of culturedMks similar to what is observed in vivo, thus making it possible toobtain several thousand platelets per Mk.

For patients undergoing ablative chemotherapy or those with certaingenetic disorders, there is a need for hematopoietic stem and progenitorcell (HSPCs) transplantation to reconstitute the hematopoietic systemthat is destroyed by chemotherapy. The HSPCs are either autologous(collected from the patient prior to chemotherapy) or from a matcheddonor or from umbilical cord blood. Thus, there is a large need of HSPCsfor such therapies and any processes that reduces the number of HSPCsneeded for transplantation would have a huge impact on transplantation.Finally, there is a large need to develop reliable gene-therapytechnologies that would allow the modification of a patient's HSPCs inorder to correct genetic disorders.

The disclosed invention has great translational potential for thedevelopment of transformational technologies in transfusion medicine,stem-cell transplantation and gene and related therapies involvingHSPCs.

Mks derive from HSPCs in the BM, and as they mature they migrate to theendothelial lining of BM sinusoids where they extend PPTs through gapsof the endothelium into circulation. Mks encounter biomechanicalstresses as they deform to penetrate gaps of the sinusoid wall, andshear stresses upon exposure to blood flow. Upon entering circulation,Mk fragments or whole Mks are exposed to shear stresses of a broad rangeand duration in different parts of circulation. Released Mk fragmentsmature into platelets in circulation, while released whole Mks areeventually captured in the pulmonary vasculature where they give rise toplatelets.

Following pioneering visualization studies [1] identifying aphysiological shear-stress range of 1.3-4.1 dyn/cm² for plateletbiogenesis in the BM, a role for shear stress was supported by an invitro study [4] demonstrating that a high shear rate (1800 s⁻¹;corresponding to ca. 16 dyn/cm², almost 4-fold higher than the upperphysiological limit in the BM) accelerates (but was not shown if itincreases) PPT formation and platelet biogenesis from cultured, matureMks. Yet, the cellular events underlying the effects of mechanicalstresses on Mk maturation and platelet biogenesis remain largelyunexplored. Shear and other biomechanical stresses affect different celltypes in biologically multifaceted and complex ways. E.g., shear stressis an important differentiation signal for embryonic stem cells,endothelial progenitor cells circulating in peripheral blood, andmesenchymal stem cells. Many cellular processes are affected by shearforces, including the cell cycle, migration, apoptosis anddifferentiation.

Cell-derived microparticles (MPs also known as microvesicles; MVs) aremembrane-bound vesicles with diameter from 100 to 1000 nm and can bederived from almost all types of cells by direct budding from plasmamembrane. They are different from exosomes (<100 nm), which originatefrom multivesicular bodies through cell exocytosis.

MP generation is always associated with cell growth, and some type ofstimulus which could be cell activation or some kind of stress stimulus.Different stimuli have been reported for different cell types for thegeneration of MPs. A very wide range of stimuli can induce cells toproduce MPs in vitro, including different types of physicochemicalstress (e.g., shear, hypoxia and oxidative stress), physiologicalactivators (e.g., thrombin, Fas ligand and tumor necrosis factor alpha)and non-physiological agonists (e.g., lipopolysaccharide and calciumionophore A23187). Upon stimulation, cells undergo activation orapoptosis and different amount of MPs are released from different typesof cells. The released MPs are heterogeneous with respect to theirsurface marker expression, membrane phospholipid composition, andinternal RNA and protein repertoires as well as their biologicalactivities even when they are from the same parent cells but generatedunder different stimulation conditions.

There is no universal mechanism leading to MP release. Cytosolic Ca⁺elevation, oxidative stress, cytoskeleton reorganization, caspaseactivation and lipid raft are involved in MP biogenesis.

MPs exert various and diverse biological effects on target cells, andthis variety depends largely on the variety of bioactive moleculescarried by MPs. It has been shown that surface markers, proteins, mRNA,microRNA, DNA or even phospholipid can act as signaling molecules insidetarget cells. For example, MPs from G-CSF-activated primary monocytes orPMA-stimulated THP-1 monocytic cells induced differentiation ofmonocytes into macrophages and this process was mediated by miR-223transfer [2]. Under many conditions, the biological function of MPs isnot mediated simply by one type of signaling molecules. Severalmechanisms, including MP attachment to cells, direct fusion andendocytosis, have been proposed and examined by studies to explainuptake process of MPs by target cells.

The biological function of MPs during intercellular communication isdependent on the interaction of MPs with and subsequently transmissionof signaling to target cells. Three different types of MP-cellinteraction have been demonstrated. Binding of MPs to cells is the firststep of interaction and several studies have demonstrated this processcould be target-specific. For example, platelet-derived microparticles(PMPs) could transfer tissue factor to monocytes but not to neutrophilsthough PMPs could adhere to both types of cells through CD62P. In somecases, MP binding is sufficient to alter the fate of target cellsthrough activation of receptors on the target cells via thecorresponding ligands present on the MP surface. MPs from endothelialcells, monocytes, platelets or human blood could bind to plateletsthrough exposed phosphatidylserine on MPs and its receptor CD36 onplatelets, and this CD36-dependent binding event augmented plateletactivation in response to low dose of ADP [3]. In some cases, MPs aretaken up by target cells following binding through two distinctmechanisms: membrane fusion and endocytosis. Both mechanisms could leadto membrane receptor transfer and internal “cargo” discharge into thetarget cells. PMPs were internalized by human brain endothelial cellsthrough active endocytosis and this led to modified endothelial cellphenotype and functions.

Chinese patent CN 104195107A discloses the application of microvesiclesfrom activated platelets in megakaryocytic differentiation of stemcells. Part of the present invention discloses the use of megakaryocyticmicroparticles in inducing megakaryocytic differentiation ofhematopoietic stem cells and in ex vivo platelet production. CN104195107A uses microvesicles from activated platelets, and thus theseare NOT MkMPs. Equally important, although they discuss that the MPsthey produce from activated platelets enhance the megakaryocyticdifferentiation of HSPCs, they add thrombopoietin, the primary cytokinethat induce megakaryocytic differentiation, in the culture medium forstem cells in the invention (CN 104195107A) while the present inventiondoes not require thrombopoietin. Thus, the present invention is distinctfrom this invention in CN 104195107A. One will see below that thepresent invention uses MkMPs from megakaryocytes that we have shownabove are very different from the MPs from activated platelets, which weshow that in the absence of TPO cannot induce megakaryocyticdifferentiation of HSPCs as shown in FIG. 9 and FIG. 12.

SUMMARY OF THE INVENTION

The present invention relates to shear stress enhancing DNA synthesis,polyploidization and apoptosis of immature megakaryocytic cells (Mkcells), and increases the formation of platelet-like particles (PLPs),pro/preplatelets (PPTs), and Mk microparticles (MkMPs). In addition,shear accelerates DNA synthesis of immature Mks in an exposure-time andshear stress level dependent manner Both early (phosphatidylserineexposure) and late (caspase-3 activation) apoptotic events were enhancedby shear stress. Inhibition of caspase-3 reduced the number ofshear-induced PLP/PPT and MkMP formation. Exposure to physiologicalshear enhances PLP/PPT formation by up to 10.8 fold. PLPs generatedunder shear flow displayed improved functionality as assessed by CD62Pexposure and fibrinogen binding. MkMP generation was dramaticallyenhanced (up to 47 fold) by shear stress. Significantly, coculture ofMkMPs with hematopoietic stem and progenitor cells (HSPCs) promoted HSPCdifferentiation to mature Mks synthesizing alpha- and dense-granules andforming proplatelets in the absence of exogenous thrombopoietin, thusidentifying, for the first time, a novel and unexplored potentialphysiological role for MkMPs. Through light, transmission or scanningelectron microscopy analysis, it is seen that MkMPs could fuse and thentransfer internal “cargo” into HSPCs. We show that RNase treatmentdestroys the endogenous RNA (as shown by decreased effect of MkMPs onHSPCs) and that MkMPs can be loaded desirable molecules for delivery toHSPCs with effectiveness and specificity.

The present invention discloses that PLP/PPT particles as well as MkMPsgenerated from various cell types (HPSCs, embryonic or iPS cells) undermechanical stress in laminar flow or turbulent flow in mixed bioreactorscan be used in the development of autologous or allogeneic celltherapies to treat thrombocytopenias, as a substitute to platelettransfusions or to enhance HSPC transplantation. We also claim thatMkMPs can be used as a means to modify in vitro or in vivo hematopoieticstem and progenitor cells by transferring specific nucleic acids (RNA orDNA molecules) or non-nucleic acid morphogens (proteins or othermolecules) to these cells. This process can be used for gene and celltherapies based on HPSCs. It can be also used to determine whatmolecules result in desirable cell differentiation and morphogenesis ofthe targeted stem and progenitor cells aiming to achieve desirablephenotypes such as production of different blood cells in vivo ortrans-differentiation to other cell types.

The present invention discloses methods to generate large number ofparticles (PLPs, PPTs) from cultured megakaryocytes (Mks or Mk cells)under shear and other biomechanical forces that lead to biologicallyactive particles for platelet functions.

The present invention also discloses methods to generate large number ofMk microparticles (MkMPs) or Mk microvesicles (MkMVs) from cultured Mkcells under shear and other biomechanical forces that lead tobiologically active particles that can be used to transfer biologicalmaterial (RNA, DNA, proteins and other biological or non-biologicalcomponents or chemicals) to other cells, including hematopoietic stemand progenitor cells.

The present invention further relates to applications of MkMPs and otherparticles produced from Mk cells alone or as supplements tohematopoietic stem cells for transplantations (also known as bone-marrowtransplantations) to enable or enhance the reconstitution of thehematopoietic system.

The present invention also relates to all biological applications exvivo or in vivo of MkMPs.

The present invention also relates to methods for loading MkMPs, PLPsand PPTs with exogenous RNA, DNA, proteins and drugs for delivery totarget cells.

The present invention also relates to methods for unloading first MkMPs,PLPs, and PPTs from native RNA, DNA and select proteins and non-proteinmorphogens. These unloaded particles can be loaded subsequently withdesirable with exogenous RNA, DNA, proteins and drugs for delivery totarget cells.

The present invention also relates to methods using stirred and tubularbioreactors for producing MkMPs, PLPs, PPTs from Mk cells derived fromHSPCs or embryonic or iPS cells, but also MPs from other cell types.These bioreactors can be used with controlled levels of biomechanicalforces to maximize the production of various biological particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sketch of the flow system used to expose Mk cells to shear flowby continuously perfusing medium over Mks attached to flow slides. a,two infusion and withdraw syringe pumps; b, dual check valves; c,extracellular matrix-coated flow slide; d, medium flow; e, mediumreservoir; f, Mks.

FIG. 2. Shear stress enhances DNA synthesis of immature Mks. The BrdUpercentage of all adherent and non-adherent Mks (A-C) or Mks withdifferent ploidy classes (2N, 4N and >=8N) (D-F) upon exposure tovarious shear-stress conditions. After shear-flow application, Mks werecultured in the presence of BrdU for a total of 4 hours. Cells were thenharvested for CD41 and PI (DNA) staining and analyzed by flow cytometry.(A, D) At d8, Mks were exposed to shear stress at level of 2.5 dyn/cm²for 0 (static control), 15, 30, 60, and 120 minutes. (B, E) At d8, Mkswere exposed to shear stress at 0 (static control), 1.5, 2.5 and 4.0dyn/cm² for 30 minutes. (C, F) At d8, d10, and d12, Mks were exposed to2.5 dyn/cm² for 30 minutes. (A, B, C, F) Adherent Mks are shown on theleft side and non-adherent cells are shown on the right side of charts.(C) The white bars represent static condition and the grey barsrepresent flow condition. (D) The white bars filled with vertical linesrepresents adherent Mks with 0 minute of flow exposure; the light greybars filled with vertical lines represents adherent Mks with 15 minutesof flow exposure; the dark grey bars filled with vertical linesrepresents adherent Mks with 30 minutes of flow exposure; the white barsfilled with horizontal lines represents adherent Mks with 60 minutes offlow exposure; the light grey bars filled with horizontal linesrepresents adherent Mks with 120 minutes of flow exposure; the dark greybars filled with horizontal lines represents non-adherent Mks with 0minute of flow exposure; the black bars represents non-adherent Mks with120 minutes of flow exposure. (E) the white bar filled with verticallines represent adherent Mks exposed to shear stress at level of 0dyn/cm²; the white bar filled with horizontal lines represent adherentMks exposed to shear stress at level of 1.5 dyn/cm²; the light grey barfilled with vertical lines represent adherent Mks exposed to shearstress at level of 2.5 dyn/cm²; the light grey bar filled withhorizontal lines represent adherent Mks exposed to shear stress at levelof 4.0 dyn/cm²; the dark grey bar filled with vertical lines representnon-adherent Mks exposed to shear stress at level of 0 dyn/cm²; the darkgrey bar filled with horizontal lines represent non-adherent Mks exposedto shear stress at level of 2.5 dyn/cm²; (F) The bars filled withvertical lines represent static condition and the bars filled withhorizontal lines represent flow condition; the white bars represent Mksat d8; the light grey bars represent Mks at d10; the dark grey barsrepresent Mks at d12. Error bars indicate standard error of mean (SEM)of 3 biological replicates. *P<0.05; P<0.05 compared to correspondingstatic control; ns, not significant.

FIG. 3. CD41+% and ploidy distribution of Mks exposed to shear stressversus static condition. (A-C) % CD41+ of cells. (D-F) ploiddistribution of Mk cells. (A, D) At d8, Mks were exposed to shear stressat level of 2.5 dyn/cm2 for various times: 0, 15, 30, 60, and 120minutes. (B, E) At d8, Mks were exposed to shear stress at variouslevels: 0, 1.5, 2.5 and 4.0 dyn/cm2 for 30 minutes. (C, F) At d8, d10,and d12, Mks were exposed to shear stress at level of 2.5 dyn/cm2 for 30minutes. (A, B, C, F) Adherent Mks are shown on the left side andnon-adherent cells are shown on the right side of charts. (C) The whitebars represent static condition and the grey bars represent flowcondition. (D) The white bar filled with vertical lines representsadherent Mks with 0 minute of flow exposure; the light grey bar filledwith vertical lines represents adherent Mks with 15 minutes of flowexposure; the dark grey bar filled with vertical lines representsadherent Mks with 30 minutes of flow exposure; the white bar filled withhorizontal lines represents adherent Mks with 60 minutes of flowexposure; the light grey bar filled with horizontal lines representsadherent Mks with 120 minutes of flow exposure; the dark grey bar filledwith horizontal lines represents non-adherent Mks with 0 minute of flowexposure; the black bar represents non-adherent Mks with 120 minutes offlow exposure. (E) The white bars filled with vertical lines representadherent Mks exposed to shear stress at level of 0 dyn/cm²; the whitebars filled with horizontal lines represent adherent Mks exposed toshear stress at level of 1.5 dyn/cm²; the light grey bars filled withvertical lines represent adherent Mks exposed to shear stress at levelof 2.5 dyn/cm²; the light grey bars filled with horizontal linesrepresent adherent Mks exposed to shear stress at level of 4.0 dyn/cm²;the dark grey bars filled with vertical lines represent non-adherent Mksexposed to shear stress at level of 0 dyn/cm²; the dark grey bars filledwith horizontal lines represent non-adherent Mks exposed to shear stressat level of 2.5 dyn/cm²; (F) The bars filled with vertical linesrepresent static condition and the bars filled with horizontal linesrepresent flow condition; the white bars represent Mks at d8; the lightgrey bars represent Mks at d10; the dark grey bars represent Mks at d12.Error bars indicate standard error of mean (SEM) of 3 biologicalreplicates. *P<0.05.

FIG. 4. Shear stress promotes phosphatidylserine (PS) externalizationand caspase-3 activation, the early and late events of apoptosis.Caspase-3 is involved in shear-stress enhanced PPTs and PLPs formation.(A) Percent of Annexin V⁺ Mks at d8 and d10 Mks after shear-flowapplication at 1 dyn/cm² for 2 hours versus static control. Adherent(left side) and non-adherent (right side) Mks were analyzed separately.The white bars represent Mks under static condition and the grey barrepresents Mks under flow condition. (B) Percent of Annexin V⁺ d10 Mksexposed to 2.5 dyn/cm² for 0 (static control), 15, 30, 60, or 120minutes. Adherent (left side) and non-adherent (right side) Mks wereanalyzed separately. (C) Correlation between caspase-3 activation andproplatelet (PPT) formation of Mks under static culture conditions.X-axis: caspase-3 activation level defined as the ratio of meanfluorescence intensity (MFI) of active caspase-3 over IgG control.Y-axis: percent of Mks bearing PPTs. (D) MFI of active caspase-3 per μm²of adherent d10 (left side) and d12 (right side) Mks from differentdonors (don) after shear-flow exposure at 1.0 dyn/cm² for 2 hours versusstatic control. The white bars represent Mks under static condition andthe grey bar represents Mks under flow condition. The ratio of MFI ofcaspase-3 of Mks under shear-flow conditions over static control isindicated above the bars. Under shear flow, MFI values for caspase-3activation were well above the MFI for isotype control, so there was noneed to correct for isotype control. (E, F) At d10 and d12, DMSO(vehicle control, white bars) or z-VAD.fmk (pan-caspase inhibitor, greybars) or z-DEVD fmk (caspase-3 inhibitor, black bars) treated Mks wereexposed to shear flow at 2.5 dyn/cm² for 0.5 hour. After shear-flowexposure, PPTs and PLPs were harvested and counted. The number of PPTs(E) or PLPs (F) from one slide of Mks exposed to shear flow wasnormalized by number of PPTs or PLPs on a slide under static conditions,and the resulting ratios are plotted. Error bars indicate SEM of 3-4biological replicates in panel (A, B, E and F) and 6˜10 different imagesin panel (D). **P<0.01; *P<0.05; ns, not significant.

FIG. 5. Caspase-3 is activated during Mk differentiation under staticculture conditions. Representative image showing that both round Mkswithout PPTs and Mks with PPTs (arrow) are positive of active caspase-3at d10 and d12 under static culture conditions. The scale bar represents50 μm.

FIG. 6. Shear stress enhances the production of pre-/pro-platelets(PPTs) and platelet-like particle (PLP). PLPs generated under shear flowdisplay enhanced in vitro functional activity. (A) A mature andpolyploid Mk displays extensive PPTs in static culture at d12. Scalebar: 50 μm. (B-D) Mks at d12 were exposed to a shear flow at 1 dyn/cm²for 2 hours or 4.0 dyn/cm² for 0.5 hour. Both adherent and non-adherentcell fragments were analyzed post shear exposure. (B) Numbers of PLPsand PPTs per slide post shear exposure versus static control; the whitebars represent samples from static condition; the grey bars representsamples from flow condition with shear stress at 1 dyn/cm² for 2 hours;the black bars represent samples from flow condition with shear stressat 4 dyn/cm² for 0.5 hour. Two functionality assays, CD62P exposure (C)and fibrinogen binding (D), were performed on the harvested PLPs,demonstrate enhanced activity for PLPs generated under shear flow. (C,D) The white bars represent PLPs without thrombin stimulation and thegrey bars represent PLPs stimulated with 3 U/mL thrombin. Error bars inpanel (B-D) indicate SEM of 3-4 biological replicates. *P<0.05;**P<0.01.

FIG. 7. Fragmentation of Mks by shear stress. (A) A representativefluorescent image of nude nuclei (indicated by white arrow) on slides ofcultured Mk cells. The scale bar indicates 50 μm. (B, C) Mks generatedfrom culture of CD34⁺ cells from different donors (don) were exposed toshear flow at 1 dyn/cm² for 2 hours at d10 (left side) and d12 (rightside) and were then stained with phalloidin (F-actin) and propidiumiodide (DNA) for analysis. The percent of nude nuclei of the totalnumber of nuclei (B) and the mean area (μm²) of a single Mk (C) weremeasured from fluorescent images. (B, C) The white bars representsamples from static condition and the grey bars represent samples fromflow condition. Error bars indicate SEM of 6 to 10 different images.*P<0.05; **P<0.01.

FIG. 8. Shear stress exposure results in dramatically enhancedgeneration of MkMPs through activation of caspase-3. (A) d10 and d12 Mkswere exposed to medium flow at a shear stress of 2.5 dyn/cm² for 0.5hour. Whole cells were removed and the same amount of samples, asassessed by internal microbeads control, from slides exposed to shearflow and static control were analyzed by flow cytometry. PPTs werelocated on the high gate, PLPs in the middle gate, and MkMPs in the lowgate. The number of particles in each gate is displayed below each gate.(B) MkMPs were smaller than microbeads of 1.34 μm diameter. (C)Expression of CD41 and CD42b on MkMPs from d12 Mk cells. (D) CD62Pexpression and the corresponding IgG control of CD41⁺ MPs from d12 Mkcells. (E) The relative number of CD41⁺ microparticles generated fromd10 or d12 Mk cells either under static conditions (white bars) or uponapplication of shear flow (grey bars) at 2.5 dyn/cm² for 0.5 hour (d10and d12) or 2 hours (d10). (F) At d10 and d12, DMSO (vehicle control,white bars) or z-VAD fmk (pan-caspase inhibitor, grey bars) orz-DEVD.fmk (caspase-3 inhibitor, black bars) treated Mks were exposed toshear flow at 2.5 dyn/cm² for 0.5 hour. After shear flow exposure, thenumber of isolated MkMPs was measured by flow cytometry. The number ofMkMPs from one slide of Mks under shear flow was normalized by thenumber of MkMPs on a slide maintained under static culture conditions,and the resulting ratios were plotted. Error bars indicate SEM of 3biological replicates. *P<0.01 in panel (E) and *P<0.05 in panel (F).

FIG. 9. MkMPs promote Mk differentiation of CD34⁺ cells and HPCs. (A)Representative graph of flow cytometry ploidy analysis of cells fromvarious coculture conditions at d8. (i) CD34⁺ cells were cocultured withor without MkMPs starting at d0. (ii) HPCs from d3 culture without (toppanel) or with (bottom panel) TPO were cocultured without or with MkMPs,PMPs from d3 to d8. The same fraction of cells from each sample wasanalyzed. (B, C) At d0, 60,000 CD34⁺ cells were cocultured with (greybars) or without (vehicle control, white bars) MkMPs in a medium withoutTPO. The numbers (B) of total cells and Mks with 2N, 4N and >=8N ploidywere counted at d8. Some Mks started to form PPTs at d9 of coculture(C). (D-G) At d3, 60,000 HPCs from culture with (D, F) or without (E, G)TPO were cultured in TPO-free medium with or without the addition ofMkMPs or PMPs. Total cells (F, G) and Mks (D, E) with 2N, 4N and >=8Nploidy were counted at d8. (D, E) The white bars represent vehiclecontrol culture; the grey bars filled with horizontal lines representcoculture with PMPs generated by thrombin stimulation; the grey barsfilled with vertical lines represent coculture with PMPs generated bycalcium ionophore A23187 stimulation; the black bars represent coculturewith MkMPs. (F, G) VC=vehicle control culture; PMP(T)=coculture withPMPs generated by thrombin stimulation; PMP(A)=coculture with PMPsgenerated by calcium ionophore A23187 stimulation. Error bars indicateSEM of 3-4 biological replicates. *P<0.01; ns, not significant.

FIG. 10. Mks generated from MkMP coculture display characteristic PPTstructures and synthesize both alpha- and dense-granules. CD34⁺ cellswere cocultured with MkMPs starting at d0. (A) At d11, cells werestained for beta 1 tubulin (i, TUBB1), vWF (ii) and serotonin (iii andiv, 5-HT) to visualize PPT structures, alpha-granules anddense-granules, respectively. Panels (iii) and (iv) displaying serotoninstaining of both cells with a nucleus (panels iii) and a nuclearcellular fragments (PPTs; panels iv) to demonstrate the development ofearly development of dense-granules in cells prior to fragmentation.Scale bar: 50 μm in panel (i, ii) and 20 μm in panel (iii, iv). (B) TEMthin section of a Mk from d11 of the coculture. IMS: InvaginatedMembrane System; N: Nucleus; G: Granules.

FIG. 11. Characterization of megakaryocyte-derived MPs (MkMPs). (A)CD62P expression and concentration of CD41⁺ MPs in Mk culture from d8 tod12. At d7, enriched Mk cells were cultured at fixed concentration of200 k/mL. From d8 to d12, 100 μL cell culture medium was harvested everyday for CD41 and CD62P analyses by flow cytometry. MP concentration wascounted by flow cytometry using internal microbeads as control. The linewith squares represent CD62P⁻ level and the line with circles representsCD41⁺ MP concentration. Error bar represents standard error of mean(SEM) of 3 biological replicates. (B) Representative size distributionhistogram of MkMPs (line A) from d12 cell culture analyzed by flowcytometry using microbeads with diameter 0.88 μm (line B) and 1.34 μm(line C) as internal size standards. Representative TEM (C) and SEM (D)micrographs of MkMPs from d12 cell culture.

FIG. 12. MkMPs promote Mk differentiation of the primitive CD34⁺Lin⁻stem cells. The primitive Lineage⁻ cells were enriched from CD34⁺ cellsand cocultured with MkMPs at concentration of 10 MkMPs/cell for 8 days.(A) The numbers of Mks with different ploidy levels (2N, 4N and >=8N) inthe control culture and the MkMP coculture at d8. (B) The numbers ofCD34⁺ cells and total cells in the control culture and the MkMPcoculture at d8. The white bars represent vehicle control culture andthe grey bars represent MkMP coculture. Error bar represents SEM of 3biological replicates. **, P<0.01.

FIG. 13. Representative CD41 expression and ploidy analyses of MkMPcoculture with MSCs, HUVECs or granulocytes. Human MSCs (passage 2-4),HUVECs (passage 3-5) and CD34⁺ cell-derived granulocytes were coculturedwith MkMPs at the concentration of 10 MPs/cell for 8 days beforeharvested for CD41 expression and ploidy analyses by flow cytometry. Theresults represent two biological replicates. The top gates representCD41⁺ cells (Mks) and the bottom gates represent CD41⁻ cells.

FIG. 14. RNase treatment reduces the inducing effect of MkMPs on HSCs.CD34⁺ cells were cocultured with MkMPs (10 MkMPs/cell) treated with orwithout RNase (RNase A/T1 cocktail or RNase ONE) for 8 days beforeharvested for ploidy analysis by flow cytometry. (A) The cell numbers ofMks with different ploidy levels (2N, 4N, >=8N) in various cell culturesat d8. (B) The numbers of total Mks and total cells in various cellcultures at d8. The white bars represent vehicle control culture. Theblack bars represent coculture with MkMPs without treatment. The greybars filled with horizontal lines represent coculture with MkMPs withRNase A/T1 treatment. The grey bars filled with horizontal linesrepresent coculture with MkMPs with RNase ONE treatment. Error barrepresents SEM of 4 biological replicates. *, P<0.05; **, P<0.01.

FIG. 15. Kinetics of MkMP binding to cells. MkMPs were stained with dyeCFDA-SE and then cocultured with d3 hematopoietic progenitor cells(HPCs). At the time as indicated, some cells were harvested for analysisof mean fluorescence intensity (MFI) of CFDA-SE by flow cytometry. AllCFDA-SE MFI at different time points were normalized to MFI at 1 hourtime point. The line with circles represents vehicle control sample andthe line with triangles represents MkMP coculture. Error bar representsas SEM of 3 biological replicates.

FIG. 16. Uptake of MkMPs by HPCs is through endocytosis while PMPs arenot taken up by HPCs. MkMPs and PMPs were stained with CFDA-SE (Green)dye and then cocultured with HPCs for 3-5 hours. Fluorescent andDifferential Interference Contrast (DIC) images were collected usingconfocal microscopy. (A) Vehicle control culture. (B, C) Two images ofthe MkMP coculture (˜4 hours). Scale bar, 20 μm.

FIG. 17. Uptake of MkMPs by HPCs is through direct fusion as shown byconfocal microscopy. MkMPs were stained with CFDA-SE (Green) dye andthen cocultured with HPCs for 3-5 hours. Fluorescent and DifferentialInterference Contrast (DIC) images were collected using confocalmicroscopy. (A) Three images of the MkMP coculture demonstrate CFDA-SEdye gradient inside the cells (red arrow). (B) CFDA-SE dye intensityprofiles of the cell #1 and #2 in panel (A) along black arrows. Scalebar, 20 μm.

FIG. 18. Uptake of MkMPs by HPCs is through direct fusion as shown byscanning electron microscopy. HPCs were cocultured with MkMPs for 3 and5 hours and examined using scanning electron microscopy. (A)Representative electron micrographs demonstrate the 4 gradual stagesthrough which MkMPs (black arrow head) were fused into cells. Scale bar,1 μm. (B) The percentages of the MkMP-cell interaction at each stageafter 3 (white bars) and 5 (grey bars) hours of coculture. Error barsindicate SEM of 2 biological replicates.

FIG. 19. Uptake of MkMPs by HPCs is through direct fusion as shown bytransmission electron microscopy. HPCs were cocultured with MkMPs for3˜5 hours and examined using transmission electron microscopy. (A) OneMkMP interacted with one cell and displayed lamellipodia-like structure(white arrow). (B) Two MkMPs interacted with one cell. The membranebetween the cell and the MkMP at the bottom was diminished.

FIG. 20. CHRF-derived MPs (CMPs) induce Mk differentiation ofhematopoietic stem cells (HSCs). HSCs were cocultured with or without(vehicle control) CMPs at the concentration of 50 MPs/cell for 8 dayswithout thrombopoietin in culture medium. Cells were harvested for CD41and DNA staining and analyzed by flow cytometry. (A) Representative flowanalysis of ploidy and CD41 expression of cells from vehicle controlculture and CMPs coculture. (B) Cell numbers of Mks with differentlevels of ploidy. The white bars represent vehicle control culture andthe grey bars represent coculture with CMPs. Error bars indicatestandard error of mean (SEM) of 3 biological replicates.

FIG. 21. The effect of RNase treatment on CHRF-derived MPs (CMPs)inducing Mk differentiation of hematopoietic stem cells (HSCs). CMPswere first treated with or without RNase A/T1 or RNase ONE for 1 hr at37° C. RNase inhibitor, SUPERase-In, were added to stop RNase reaction.HSCs were then cocultured with CMPs or with RNase-treated CMPs orwithout CMPs at the concentration of 50 MPs/cell for 8 days. The whitebars represent vehicle control culture; the black bars representcoculture with untreated CMPs; the grey bars filled with horizontallines represent coculture with CMPs treated with RNase A/T1; the greybars filled with vertical lines represent coculture with CMPs treatedwith RNase ONE. Cells were analyzed of ploidy and CD41 expression byflow cytometry. Cell numbers of Mks with different levels of ploidy wereshown in figure. Error bars indicate standard error of mean (SEM) of 3biological replicates.

FIG. 22. Flow cytometry analysis of MPs loaded with plasmid DNAs usingelectroporation. Plasmid DNA, pmaxGFP, were conjugated with redfluorescent dye Cy5. Then plasmids pmaxGFP were loaded into MkMPs andCMPs using electroporation. The resulting MkMPs and CMPs were analyzedby flow cytometry. Electroporation was performed using AMAXANucleofector™ II Device. The inserts represent MP withoutelectroporation.

FIG. 23. Brief procedure of loading pmaxGFP DNA by electroporation.

FIG. 24. In this drawing, “Modification” refers to unloading RNAs fromMkMPs and/or reloading MkMPs with the desirable RNAs, DNAs, proteins orother therapeutic drugs. HSPCs, hematopoietic stem and progenitor cells;Mks, megakaryocytes; PPT, pro/preplatelets; PLPs, platelet-likeparticles; MkMPs, megakaryocytic microparticles.

DETAILED DESCRIPTION

While the present disclosure may be susceptible to embodiments indifferent forms, and herein various embodiments will be described indetail with the understanding that the present description is to beconsidered an exemplification of the principles of the disclosure and isnot intended to be exhaustive or to limit the disclosure to the detailsof construction and the arrangements of the components set forth in thefollowing description or illustrated in the drawings.

Methods Used for the Disclosure and Enablement of the Invention

Materials and Proteins:

All chemicals were obtained from Sigma-Aldrich or otherwise indicated.Recombinant human interleukin 3 (IL-3), IL-6, IL-9, IL-11, stem cellfactor (SCF), granulocyte colony-stimulating factor (rhG-CSF) andthrombopoietin (TPO) were from purchased from PeproTech Inc. Purifiedhuman von Willbrand Factor (vWF, Factor VIII free) was from HaematologicTechnologies. Human fibrinogen for coverslip coating was from InnovativeResearch. Alexa Fluor® 647-conjugated fibrinogen for plateletfunctionality assays was from Life Technologies. Phycoerythrin(PE)-conjugated Annexin V was from BD Bioscience. Human thrombin wasfrom Sigma-Aldrich. Human thrombin was from Sigma-Aldrich. Size standardfluorescent beads (0.22, 0.45, 0.88 and 1.34 μm) and AccuCountfluorescent particles (5.1 μm) were from SpheroTech.

Antibodies:

Fluorescein isothiocyanate (FITC)-conjugated anti-CD41a (GPαIIb),PE-conjugated anti-CD42b (GPIbα), PE-conjugated anti-CD62P,allophycocyanin (APC)-conjugated anti-BrdU (BrdU APC flow kit),APC-conjugated anti-CD34, PE-conjugated CD11b and APC-conjugatedanti-CD235a antibodies were from BD Bioscience. Anti-active caspase-3antibody (ab13847), anti-human β1 tubulin antibody (ab96008), anti-humanvWF antibody (ab9378) and anti-serotonin antibody (ab66047) were allfrom Abcam. The secondary antibodies, Alexa Fluor® 488-conjugatedanti-rabbit IgG antibody and anti-goat IgG antibody, were from LifeTechnologies. FITC-conjugated CD41 antibody for CD41⁺-cell enrichmentand platelet functionality assays was from Beckman Coulter.

Megakaryocytic Cultures:

Frozen G-SCF mobilized peripheral blood CD34⁺ cells were obtained fromthe Fred Hutchinson Cancer Research Center. CD34⁺ cells were culturedusing the protocol previously described. Briefly, from day 0 (d0) to d5,cells were cultured in Iscove modified Dulbecco medium (IMDM, GlutaMax™;Life Technologies), pH 7.2, supplemented with 20% BIT9500 (StemcellTechnologies), 100 ng/mL rhTPO, 100 ng/mL rhSCF, 2.5 ng/mL rhIL-3, 10ng/mL rhIL-6, 10 ng/mL rhIL-11 and 1 μg/mL human low density lipoprotein(hLDL), at 37° C. in fully humidified incubator under 5% CO₂ and 5% O₂.Then from d5 to d7, culture medium was changed to IMDM, pH 7.4,supplemented with 20% BIT9500, 100 ng/mL rhTPO, 100 ng/mL rhSCF, 10ng/mL rhIL-3, 10 ng/mL rhIL-9, 10 ng/mL rhIL-11 and 1 μg/mL hLDL, and O₂level was increased to 20%. At d7, CD61⁺ cells (Mks) were enriched usinganti-CD61 magnetic microbeads (Miltenyi Biotec) and LD magnetic columns(Miltenyi Biotec). After enrichment, Mks (CD41⁺ purity>90%) werecultured in IMDM, pH 7.6, supplemented with 20% BIT9500, 100 ng/mLrhTPO, 100 ng/mL rhSCF, 1 μg/mL hLDL and 6.25 mM nicotinamide. From d8to d12, CD41 and CD62P expression and concentration of microparticles(MPs) in cell culture were measured by flow cytometer (FACSAria II, BDBiosciences) using AccuCount fluorescent particles as internal control.

Shear-Stress Experiments: Exposure of Mk Cells to Shear Flow:

Rectangular flow slides (μ-Slide 1^(0.6) Luer, ibidi USA) were coatedwith 50 μg/mL vWF, and ca. 300,000 cultured Mks were seeded into eachslide. Mks on slides were cultured overnight (21 hours) before beingexposed to shear flow. Medium (IMDM supplemented with 10% BIT9500, 50ng/mL TPO, 50 ng/mL rhSCF, 0.5 μg/mL hLDL and 6.25 mM nicotinamide) wasperfused over Mks on slides by two syringe pumps (Dual NE-4000 pump; NewEra Pump Systems) to achieve the desirable shear-stress level. For BrdUincorporation assays, the medium was supplemented with 10 μM BrdU (BD).During shear flow, some Mks were detached from the slide surface andreleased into the circulating medium. These are considered asnon-adherent Mks. Adherent Mks were harvested for analysis usingnon-enzymatic cell dissociation buffer (Sigma-Aldrich). In someexperiments, adherent Mks were fixed with 2% paraformaldehyde directlyon slides and processed for immunofluorescence analysis. In someexperiments, Mks were treated with caspase inhibitors, 10 μM z-VAD.fmk(Bachem) or 10 μM z-DEVD.fmk (Bachem) starting on d9. Inhibitor-treatedMks were seeded into flow slides at d9 or d11, were exposed to shearflow (2.5 dyn/cm² for 0.5 hour) in medium supplemented with the sameinhibitor, and were harvested for PPT, PLP and CD41⁺ microparticlecounting.

DNA Synthesis Assay:

DNA synthesis was assessed using a BrdU APC flow kit (BD Bioscience).After exposure to shear flow for the indicated time, Mks were culturedfor additional time period to a total 4 of hours with BrdU in the mediumbefore harvesting for analysis. Cells from static cultures were treatedthe same way and served as control

Annexin V Assay:

After shear flow application or static control, cells were harvestedimmediately and stained with FITC-anti-CD41a antibody and PE-Annexin Vfor flow-cytometric analysis.

Immunofluorescent Staining:

For β1 tubulin staining, cells were fixed and permeabilized using 1%glutaraldehyde and 0.1% Triton® X-100 (Sigma-Aldrich) in PHEM buffer (60mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 6.9). Then, cells werequenched in 1-2 mg/mL sodium borohydride before blocking. For otherstaining, cells were fixed with PFA and permeabilized with Triton X-100.After blocking with BSA (Fisher Scientific) together with normal goat ordonkey serum (MP Biomedicals), primary antibodies (active caspase-3, β1tubulin, vWF or serotonin) or corresponding isotype controls wereapplied to cells overnight at 4° C., followed by incubation with thesecondary antibody conjugated with Alexa Fluor® 488 at room temperaturefor 1 hour. F-actin and DNA were stained with Alexa Fluor®568-phalloidin (Life Technologies) and TO-PRO®-3 (Life Technologies),respectively. Fluorescent images were collected via a multiphotonconfocal microscope (Zeiss 510 NLO). Mean fluorescent intensity (MFI) ofactive caspase-3 and the average area for a single Mk were quantifiedusing Velocity® Image Analysis Software (Perkin Elmer).

Isolation of PLPs:

Large cells were excluded from PLP preparations by centrifugation at150× g for 10 minutes. PLPs were then pelleted by centrifugation at1000× g for 10 minutes from the PLP-enriched supernatant. After onewash, PLPs were resuspended in Tyrode's buffer and used inplatelet-stimulation assays. The number of PLPs and PPTs per slide wasmeasured using a Multisizer™ 3 Coulter Counter (Beckman Coulter).

Platelet-Stimulation Assays: CD62P Exposure and Fibrinogen Binding:

These assays were carried out as described, whereby CD62P expression andfibrinogen binding were measured by flow cytometry.

Preparation of Human Platelet and Platelet-Derived Microparticles(PMPs):

Blood for isolation of human platelets was collected by venipuncturefrom adult human volunteers after providing written informed consent asapproved by the Institutional Review Board at University of Delaware(IRB protocol #190471-3). Blood was collected into a 60-cc syringecontaining ACD (trisodium citrate, 65 mM; citric acid, 70 mM; dextrose,100 mM; pH 4.4) at a ratio of 1:6 parts ACD/blood. Anticoagulated bloodwas spun by centrifugation at 250× g and the supernatant containingplatelet rich plasma (PRP) was then pelleted at 750× g (10 minutes),washed once in HEN buffer (10 mM HEPES, pH 6.5, 1 mM EDTA, 150 mM NaCl)containing 0.05 U/ml apyrase and platelets resuspended in HEPES-Tyrode'sbuffer (137 mM NaCl, 20 mM HEPES, 5.6 mM glucose, 1 g/l BSA, 1 mM MgCl2,2.7 mM KCl, 3.3 mM NaH2PO4) at a concentration of 4×10⁸ platelets/ml inHEPES-Tyrode's buffer containing 0.05 U/ml apyrase.lmM CaC₂ was added toplatelet before activation and platelets were activated by 2 U/mL humanthrombin or 10 μM Calcium Ionophore (A23187, Sigma-Aldrich). Theplatelets were removed by centrifugation at 1000× g for 10 minutes andPMPs were harvested from supernatant washed two times using IMDM mediumby ultracentrifugation at 25,000 rpm for 1 hour, 4° C. The concentrationof PMPs was measured by flow cytometry using 1.34 μm-diametermicrobeads.

ELISA Assay for TPO:

Protein lysates and supernatants from concentrated microparticlesuspensions were analyzed using human TPO ELISA (PeproTech) according tomanufacturer's protocol. The signal was read at 405 nm on a PerkinElmerVictor 3V multilabel counter.

Isolation and Characterization of MkMPs:

For both static cultures and cultures exposed to shear flow, Mk cellswere removed from the culture medium by centrifugation (150× g for 10minutes). Following that, PLPs were removed by centrifugation at 1000× gfor 10 minutes. Particles were then washed twice in IMDM medium and wereenriched for MkMPs by ultracentrifugation (25,000 rpm for 1 hour at 4°C.; Beckman Coulter Optima Max Ultracentrifuge). CD41, CD42b and CD62Pexpression was examined by flow cytometry. Concentrations of MkMPs (andof PMPs and Mks) were measured by flow cytometry using 1.34 μmmicrobeads (Sphero Tech) as standard. For some experiments, MkMPs insupernatant were incubated with 1 U/mL RNase A/T1 cocktail (LifeTechnologies) or 10 U/mL RNase ONE™ (Promega) for 1 hour at 37° C.before enrichment.

Human Umbilical Vascular Endothelial Cells (HUVECs), Mesenchymal StemCells (MSCs) and Granulocytic Cultures:

Primary HUVECs were obtained from ATCC and cultured according to ATCCrecommendation (growth medium from ATCC: vascular cell basal mediumsupplemented with endothelial cell growth kit-VEGF). Human MSCs wereobtained from Lonza and cultured according to Lonza recommendation(growth medium: mesenchymal stem cell basal medium supplemented withMSCGM™ SingleQuots™). Human granulocytes were differentiated from humanCD34⁺ cells as previously described. Human long-term medium (HLTM) wasprepared by supplementing McCoy's 5A medium with 12.5% heat-inactivated(57° C. for 30 minutes) fetal bovine serum (Hyclone), 12.5%heat-inactivated horse serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 1%minimal essential medium (MEM) essential amino acid solution (LifeTechnologies), 1% MEM nonessential amino acid solution (LifeTechnologies), 1% MEM vitamin solution (Life Technologies), 100 mMmonothioglycerol, 10 mM HEPES, and 50 mg/mL gentamycin sulfate. CD34⁺cells were cultured in HLTM supplemented with 50 ng/mL rhSCF, 10 ng/mLrhIL-3, rhIL-6 and rhG-CSF (supplement fresh rhG-CSF every 2 days due todegradation) in fully humidified incubator under 5% CO₂ and 5% O₂. At d7of cell culture, CD15⁺ cells were enriched using MS column and CD15microbeads (Miltenyi Biotec).

Mk Ploidy Analysis:

Cells from MkMP coculture were stained with FITC anti-CD41 antibodybefore fixation by 0.5% paraformaldehyde (Electron Microscopy Sciences)and permeabilization by 70% methanol/H₂O. After RNA was degraded byRNase A (Life Technologies), DNA was stained with 100 μg/mL propidiumiodide. Analyses of CD41 expression level, cell ploidy and numbers wereperformed on flow cytometry using AccuCount fluorescent particles asinternal control.

MkMP Binding and Uptake Analysis:

MkMPs were stained with 20 μM CFDA-SE (Life Technologies) for 20 minutesat 37° C. and washed three times in IMDM medium. Then MkMPs werecocultured with HPCs from d3 Mk culture at concentration of 30MkMPs/cell for indicated time before analysis. For the first hour, thecoculture medium was 50 μL IMDM and after that coculture was diluted inIMDM supplemented with 5% BIT9500, 50 ng/mL rhSCF and 1% pen strep (LifeTechnologies). Flow cytometry was used to measure binding of MkMPs tocells. In some experiments, after 3 hours, images of coculture werecollected via confocal microscope (Zeiss 5 LIVE DUO Highspeed/SpectralConfocal, Bioimaging Center, Delaware Biotechnology Institute).

Transmission Electron Microscopy (TEM):

Cells from coculture were fixed in 2% glutaraldehyde and 2%paraformaldehyde in 0.2 cacodylate buffer overnight at 4° C., washed,postfixed within 2% osmium tetroxide for 1 hour at room temperature,followed by 4 washes in H₂O. The samples were then stained en blocovernight at 4° C. with 1% uranyl acetate. After dehydrated in a seriesof ascending acetone solutions, samples were infiltrated within n-BGEand then Quetol-NSA resin. Samples were then embed in labeled BEEMcapsules and polymerized at 60° C. for 24-48 hours. Ultrathin sectionswere prepared using a Reichert Jung Ultracut E ultramicrotome, and werecollected onto 200 mesh formvar/carbon coated copper grids. Grids werestained with 2% methanolic uranyl acetate and Reynolds' lead citrate.Transmission Electron Microscopy was performed on Zeiss Libra 120Transmission Electron Microscope and images were acquired using a GatanUltrascan 1000 CCD.

Scanning Electron Microscopy (SEM):

The d3 HPCs from Mk culture were incubated with MkMPs (10 MPs/cell) in100 μL medium for 2 or 4 hours. Then the coculture was let spread oncircle coverslip coated with 1 μg/mL human fibronectin for another hour.2% EM grade glutaraldehyde/IMDM medium was added to coverslips to fixthe cells for at least 1 hour at room temperature or overnight at 4° C.Then the samples were washed with PBS and postfixed for 1.5 hours in 1%OsO₄ in H₂O at room temperature. After rinsed with H₂O, the samples weredehydrated in a series of ascending ethanol concentrations for 10minutes in each solution. After critical-point drying by Autosamdri-815BCritical Point Dryer (Tousimis), the samples were sputter-coated withgold using a Bench Top Turbo III Sputter Coater (Denton Vacuum). Theelectron images were collected via Hitachi 54700 Field-Emission ScanningElectron Microscope (Hitachi) at a working distance of 8.5-10.5 mm andvoltage of 3.0 kV.

Statistical Analysis:

Paired Student t test of all data was performed by Minitab 16 (Minitab).Statistical significance was defined as P<0.05.

Example 1. Shear Flow Promotes DNA Synthesis and Accelerates thePolyploidization of Mks in a Largely Dose and Maturation-Stage DependentWay

Mk cells engage endomitosis as they mature and become polyploid. Wehypothesized that biomechanical forces, such as physiological shearforces, would impact DNA synthesis. To investigate this hypothesis, Mkcells from d7 of culture were seeded onto vWF-coated slides and culturedovernight before exposure to shear flow using perfusion with mediumcontaining 10 μM BrdU. We employed a validated perfusion system (FIG. 1)designed to expose cells to defined shear stress. For our experiments,we used shear in the physiological range of 1.3-4.1 dyn/cm². First, weexposed d8 Mk cells to 2.5 dyn/cm² for 0 (static control), 15, 30, 60 or120 minutes. Exposure to shear flow increased DNA synthesis of Mk cellsby up to 3-fold, and the increase was exposure-time dependent (FIG. 2A).Mks responded to shear stress quickly, certainly within 15 minutes, butafter 30 minutes, no further increase in DNA synthesis was observed.This is physiologically relevant. It has been reported that, in vivo,the time needed for trans-sinusoidal migration of murine Mk fragmentsinto the blood stream is about 30 minutes [1]. In subsequentexperiments, an exposure time of 30 minutes was used to investigate theimpact of shear-stress level on Mks. Low levels of shear stress (1.5dyn/cm²) enhanced DNA synthesis of d8 Mks by 51% compared to staticconditions (FIG. 2B). Exposure to 2.5 dyn/cm² increased DNA synthesisfurther by 37% over that of 1.5 dyn/cm², or 107% over static control(FIG. 2B). However, at 4.0 dyn/cm² (near the upper limit of thephysiological range of shear stress in the bone marrow of mammals; seeRefs. [1,9]), DNA synthesis was similar to that at 1.5 dyn/cm².

To investigate if shear flow differentially affects DNA synthesis atdifferent differentiation stages, Mks at d8, d10 and d12 were exposed to2.5 dyn/cm² for 30 minutes. Our data (FIG. 2C) show that exposure toshear flow results in increased DNA synthesis only of d8 Mk cells. Mksat d10 or d12 are more mature, thus displaying much lower DNA synthesiscompared to d8 cells. We also examined if shear affects Mks of differentploidy classes (2N, 4N, >=8N Mks) differently. DNA synthesis of eachploidy class showed trends similar to those of the total Mk population(FIGS. 2D-F). However, DNA synthesis of Mks with 4N and higher ploidyclasses increased further when 2.5 dyn/cm² was applied for 120 minutes(FIG. 2D). These data suggest that even short exposure to circulatoryshear promotes the maturation of less mature d8 Mk cells as assessed byaccelerated DNA synthesis of all ploidy classes but also by enhancedpolyploidization under some flow conditions.

We also examined the impact of biomechanical forces on non-adherentcells. In contrast to adherent Mk cells, DNA synthesis of non-adherentMk cells in these experiments was not affected compared to staticconditions (FIGS. 2A-C).

Could the effect of shear stress on DNA synthesis be due to differentialretention of adherent Mk cells because adherent Mks cells were moreactive in synthesizing DNA? Our data suggest that this is not the case.Indeed, the % CD41⁺ cells and ploidy distribution among adherent Mksunder various (stress level and duration) flow conditions and staticconditions were similar (FIG. 3). In addition, we observed decreased DNAsynthesis after the shear stress level was increased from 2.5 dyn/cm² to4 dyn/cm² (FIG. 2B), and finally, DNA synthesis of Mks at d10 and d12was not accelerated by shear stress.

We also found that Mk cells respond to higher shear stresses (up to 400dyn/cm², but more likely up to 100 dyn/cm²) such as those encountered inthe lung vasculature and systemic blood circulation (see Supplementalmaterial of ref. [9]). Mk cells are trapped in the lung vasculaturewhere they experience higher shear and normal stresses than in the bonemarrow. Mk cells are also exposed to variable (in magnitude andfrequency) shear and normal stresses, from both laminar and turbulentflows (this laminar and turbulent shear and other stresses) in the bonemarrow and in systemic blood circulation due to the pulsatile bloodflow, different blood vessel diameters and due to squeezing throughblood-vessel endothelial cells (see Refs [1, 9] and Supplementalmaterial of ref. [9]). Shear stresses can range from 0.1 to 100 dyn/cm2.Frequency of stress application can range from a few seconds, 10, 20,30, 60, 120 seconds to a few minutes, 1-10 minutes depending on locationin the blood and lung vasculatures and the trapping of Mk cells betweenother cells. The frequency of stress application may be constant or inpulsed delivery. All such biomechanical stresses of variable magnitudeand frequency can stimulate Mk cells and can lead to increased DNAsynthesis and the formation of various particles derived from Mk cellsas described in the examples below.

Shear flow is a flow of fluid in a channel that creates a shear stresson the walls of the channel or on the surfaces of objects (such as cellsor particles) in the flow channel. Shear stress, here due to fluid flow,is a mechanical stress that arises in a flow field in a channel oraround an object (such as a cell or particle) in the flow field due tothe changing fluid velocity in the channel in any cross section of thechannel or for the case of flow around an object due to the changingvelocity in the area of the flow field near the object. The shear stressis tangent to the fluid element surface. The shear stress is highest onthe wall of the channel where the velocity changes the fastest, as inthis case the cells are grown. A shear stress is also the highest nearthe surface of an object in a flow field. In a flow, the normal stressis perpendicular to the surface of the fluid element in a flow field. Alaminar shear stress arises in a laminar fluid flow, which is the fluidflow where the fluid flows in parallel thin layers, with no disruptionbetween the thin layers. Turbulent stresses arise in the complex fluidpatterns of turbulent fluid flow, in which there are eddies created inthe flow that create a chaotic pattern of fluid motion and where, incontrast to laminar flow, the fluid does not flow in orderly parallelthin layers.

Example 2. Shear Stress Promotes Phosphatidylserine (PS) SurfaceExposure on Maturing Mk Cells

As an early mark of apoptosis, previous studies in our lab and otherlabs have shown that PS becomes exposed on the extracellular side of theMk membrane when HPSCs (both human and murine) were differentiated intoMks. In this study, to differentiate human CD34⁺ cells into Mks, we useda new protocol that gives rise to functional PLPs in vitro. Using flowcytometry and microscopic analyses, we confirmed that PS is also exposedon the surface of maturing (>d8) Mks generated by this protocol (datanot shown). To investigate if shear promotes PS externalization, fluidflow at 1 dyn/cm² was applied to d8 and d10 Mks for 2 hours. Cells wereharvested immediately after the shear-flow application for analysis.Shear resulted in a significantly increased fraction (by ca. 160% and260% at d8 and d10, respectively) of adherent Mks that are Annexin V⁺,but not so for non-adherent Mks (FIG. 4A). We also examined the impactof shear exposure time on PS externalization by exposing d10 cells to2.5 dyn/cm² for 0 (static condition), 15, 30, 60 and 120 minutes. PSexternalization responded quickly to shear stress: the fraction ofadherent Mk cells that became Annexin V⁺ increased by more than 70%after 15 minutes of exposure to shear flow (FIG. 4B). PS externalizationplateaued for exposure times between 15 and 60 minutes, but increasedfurther at 120 minutes (FIG. 4B). Shear flow did not affect PSexternalization of non-adherent Mks (p>0.10) (data for 0 and 120 minutesare only shown; FIG. 4B).

Example 3. Caspase-3 Activation in Maturing Mk Cells is Accelerated byExposure to Shear Flow

It has been shown that activation of caspase-3 and 9 is required for PPTformation. Here we wanted to investigate if shear stress affectscaspase-3 activation in Mks. We chose caspase-3 as a marker of lateapoptosis to complement our Annexin V studies above, which pertain toearly apoptotic events. First, we confirmed that caspase-3 in indeedactivated during in vitro Mk maturation with our culture protocol.Caspase-3 was activated at d10 and d12 when Mks projected PPTs under ourculture protocol; active caspase-3 accumulated largely around thenucleus, but PPTs did not stain for active caspase-3 (FIG. 5). We alsoobserved a correlation between the activation level of caspase-3 in Mks(represented as the ratio of MFI of active caspase-3 over isotypecontrol) and PPT formation (FIG. 4C), which is consistent with previousstudies discussed above. Next, we investigated if shear stress enhancescaspase-3 activation in maturing Mks, notably at d10 and d12. Exposureof Mks for 2 hours to 1 dyn/cm² enhanced caspase-3 activation both atd10 and d12 (FIG. 4D) by 1.7 to 5.6 fold depending on the donor and day.Since, as discussed, caspase-3 activation is necessary for PPTformation, one mechanism by which shear stress may promote PPT formation(see below) is by enhancing caspase-3 activation. To investigate thishypothesis, Mks were treated with 10 μM z-VAD.fmk (pan-caspaseinhibitor) or z-DEVD.fmk (caspase-3 inhibitor) and were then exposed toshear flow. After flow application, PLPs (d=1-3 μm) and PPTs (d=3-10 μm)were harvested and counted. The effect of shear stress on particlegeneration was assessed using the ratio of PLP or PPT number from oneslide of Mks under flow conditions over that under static conditions. Atd10, z-VAD.fmk had no statistically significant effect on the PLP andPPT ratios, and z-DEVD fmk decreased only the PPT ratio (FIGS. 4E and4F). However, at d12, both of z-VAD fmk and z-DEVD.fmk decreased boththe PPT and PLP ratios (FIGS. 4E and 4F). These data suggest that at thed10 early maturation stage, at which time Mks were starting to projectPPTs and very few PLPs were formed, the caspase-3 inhibitor inhibitedthe effect of shear stress on PPT generation but not on PLP generation.At d12 when Mks produced more PPTs and stared to form a significantnumber of PLPs, the effect of shear on PPT and PLP generation wasattenuated by caspase inhibitors. These data suggest that caspase-3activation plays a role in the mechanism by which shear stress enhancesPPT and PLP formation.

Example 4. Shear Stress Enhances the Generation of FunctionalPlatelet-Like Particles (PLPs) as Well PLP Activity

Here we aimed to investigate and quantify the effect of shear stress onthe generation of Mk fragments with platelet-like properties at d12 whenMks had extensive PPTs (FIG. 6A). After a 2-hour exposure of adherentMks to 1 dyn/cm², 5.8 times more PLPs were formed compared to staticconditions, while exposure to 4 dyn/cm² for 0.5 hour yielded even morePLPs (ca. 10.8-fold higher than static control; FIG. 6B). Moreover, thenumber of PPTs increased by 4.1 and 7.9 fold after 2 hours of exposureto shear flow at 1 dyn/cm² and 0.5 hour of exposure to 4 dyn/cm²,respectively (FIG. 6B). These data show that, in vitro at least,exposure to physiological levels of shear results in a dramatic increasein both PLP and PPT formation from mature Mks. These data could not havebeen anticipated by the findings of the study in ref. [4], andconstitute a potent way for generating PLPs for transplantationapplication, which we claim in this application.

Next, we examined the impact of shear flow on the functionality of thegenerated PLPs. Is it possible that the fast generation of PLPs undershear flow results in lesser quality of PLPs, or perhaps the opposite?To do so, we employed two platelet-function assays, CD62P exposure andfibrinogen binding assays, both using the physiological activator: humanthrombin. For PLPs generated from Mks under 1 dyn/cm² for 2 hours, thefraction of PLPs expressing CD62P increased by almost 3-fold (from 10%to 29%) upon thrombin activation, while for PLPs generated from Mk cellsunder static conditions this fraction increased by 1.8-fold (from 9% to16%; FIG. 6C). After activation with thrombin, the % of PLPs generatedfrom Mks under shear flow (1 dyn/cm² for 2 hours) that bind fibrinogenincreased by 12-fold (from 6% to 72%), while that of PLPs from staticculture increased by ca. 6.5-fold (from 9% to 58%; FIG. 6D). The qualityof PLPs generated under 0.5-hour exposure to higher shear (4 dyn/cm²)was similar to PLPs generated under a 2-hour exposure to 1 dyn/cm²(FIGS. 6C and 6D). Taken together, these data suggest that PLPs producedfrom Mks upon exposure to shear flow have better functionality than PLPsgenerated under static conditions. To sum, exposure to shear, evenbriefly, results in dramatic increases in both the number and quality ofPLPs when compared to static controls. This finding is supporting theclaim we make to the effect that shear and generally biomechanicalforces when used for the generation of PLPs and PPTs and thus for the invitro production of platelets from cultured Mk cells will producesuperior PLPs, PPTs and platelets.

We also quantified the Mk-fragmentation outcomes aiming to illuminateand support the data of FIG. 6B. As expected, we observed a large numberof nude nuclei (FIG. 7A) remaining on the slides after exposure to shearflow at 1.0 dyn/cm² for 2 hours. The morphology of these nuclei wasround and their size similar to nuclei in intact cells, thus indicatingthat these were not apoptotic bodies. Staining for F-actin suggestedthat there was no cell cytoplasm attached to these nuclei. We quantifiedthe fraction of nude nuclei over the total number of nuclei in eachimage. Compared to slides from static cultures, the fraction of nudenuclei was higher on the slides after shear flow both at d10 and d12(FIG. 7B). In addition, we found that the mean surface area of single,intact Mks after exposure to shear flow was significantly smaller thanthat of Mks under static conditions (FIG. 7C), which shows that shearforces selectively fragment larger, more mature Mk cells. There are noprior quantitative studies on Mk-cell fragmentation under shear flow forthe generation of PLPs, PPTs, platelets and MkMPs.

Example 5. Shear Stress Dramatically Enhances the Generation ofMk-Derived Microparticles (MkMPs)

When we examined the size distribution of cell fragments released fromMks under both static and shear-flow conditions, in addition to PLPs(d=1-3 μm) and PPTs (d=3-10 μm), we found a distinct population of verysmall particles (FIG. 8A). We ran a microbead (d=1.34 μm) control toconfirm that these particles are on an average considerably smaller than1.34 μm (FIG. 8B). Mature Mks and activated platelets can give rise toMPs that are smaller than platelets. Surface staining demonstrated thatmost of these particles were CD41⁺ and CD42b⁺, but many were also CD41⁺and CD42b⁻ (FIG. 8C). In order to identify the origin of these CD41⁺particles, we examined them for CD62P expression. CD62P is expressed onPMPs but not on the MkMPs. We found that ca. 16% of the CD41⁺ MPs wereCD62P⁺, thus suggesting that most of these MPs were MkMPs deriving fromMks rather than from activated PLPs (FIG. 8D), which presumably cangenerate MPs similar to PMPs, i.e., CD62P⁺ MPs.

Cultures of Mk cells post shear exposure contained a dramatically largernumber of these MkMPs compared to those from Mks grown under staticconditions (FIG. 8A). Thus, Mk exposure to shear results in increasedMkMP formation in addition to enhanced PLPs generation. Exposure to 2.5dyn/cm² for 0.5 hour resulted in increased MkMPs generation by 24- and27-fold at d10 and d12, respectively (FIG. 8E). For d10 Mks, exposure to2.5 dyn/cm² for 2 hours resulted in a 47 fold increase in MkMPsgeneration (FIG. 8E). Next, we investigate if caspases mediate the shearstress-enhanced generation of MkMPs. As described earlier, Mks weretreated with 10 μM z-VAD.fmk or z-DEVD fmk before exposed to shearstress at d10 and d12. The ratio of the number of MkMPs from one slideof Mks under shear flow over the number of MkMPs under static conditionswas used to assess the effect of shear stress on MkMP generation. Theresults (FIG. 8F) show that only treatment with caspase-3 inhibitor(z-DEVD fmk) attenuated the effect of shear stress, suggesting thatcaspase-3 is involved in shear-enhanced MkMP generation.

Flaumenhaft et al. have shown that the CD41⁺ MPs in human plasma aremainly derived from Mks rather than activated platelets [5]. However, nomechanism for generating MkMPs was previously known, and no function forMkMPs was known either until this present set of investigations anddata. Our data support the thinking that when mature Mks enter BMsinusoids and are exposed to shear circulatory forces, numerous MkMPsare likely formed. While PMP generation from platelets on immobilizedvon Willebrand-factor (vWF) coated surfaces under high shear waspreviously shown, it was reported that vWF was necessary for thegeneration of PMPs under shear flow. These findings pertain to thegeneration of MkMPs (which are different from the PMPs) under shear flowand without the need for vWF involvement. While the cellular mechanismsleading to membrane vesiculation and MP release remain an activeresearch field, studies from PMP biogenesis suggest that PSexternalization and caspase-3 activation play an important role in MPgeneration. In our study, we found that caspase-3 activation and PSexternalization were enhanced by shear stress, thus suggesting thatshear-stress enhanced MkMP generation may be mediated by PSexternalization and caspase-3 activation. The latter is supported by thedata from the caspase-3 inhibition assays.

Example 6. Novel Biological Activity of MkMPs: Promoting MkDifferentiation of HSPCs

A physiological function for MkMPs has not been yet previously reported.This is the first study to identify the role and potential use of MkMPs.We hypothesized that a role of MkMPs might be to acceleratehematopoietic-progenitor differentiation into Mks. In early experiments,we found that MkMPs cocultured with HPCs from d5 of Mk culture fromCD34⁺ cells promoted HPC survival and Mk differentiation under serum-and TPO-free conditions. We thus examined in more detail this effectusing MPs generated from d12 Mks. We will refer to these MPs as MkMPsalthough they may contain a small fraction (ca. 16%) of CD62P⁺ MPs.MkMPs were cocultured with CD34⁺ cells in a medium without added TPO butwith 50 ng/mL rhSCF (for enhancing cell survival), and the outcomes wereexamined after 8 days of culture. In more detail, 30,000 CD34⁺ cells (orcultured HPCs from d3 culture of CD34⁺ cells with or without TPO) wereincubated with 10 MkMPs or PMPs per CD34⁺ cell or HPC in 50 μL IMDMmedium for 1 hour at 37° C. to enhance the contact between MPs andcells. After that, the cells with the MPs were diluted in 300 μL IMDMmedium supplemented with 5% BIT9500 and 50 ng/mL rhSCF and cultured at37° C. and 20% O₂. For some coculture, Lin⁺ cells (CD2⁺, CD3⁺, CD11b⁺,CD14⁺, CD15⁺, CD16⁺, CD19⁺, CD56⁺, CD123⁺, or CD235a⁺) from CD34⁺ cellsbefore coculture with MkMPs. For some coculture of MkMPs and d3 HPCs,cells were harvested after 5 hours of incubation and then processed forTEM imaging. For some cocultures, MkMPs were labeled with fluorescentdye CFDA-SE (Sigma-Aldrich) first and incubated with d3 HPCs for varioustimes before analysis by flow cytometry. For other coculture, cells wereharvested on d8 for CD41 and ploidy flow-cytometry analysis. At d9,cells in coculture were examined using multiphoton confocal microscope(Zeiss 510 NLO), and DIC (Differential Interference Contrast) imageswere collected. At d10, cells from coculture were seeded onto humanfibrinogen-coated coverslips and cultured overnight for staining for β1tubulin (TUBB1), vWF and serotonin (5-HT) at d11. Cells from vehiclecontrol were fixed first and cytospun onto coverslip using ShandonCytospin 4 (Thermo Scientific) before immunofluorescent staining. Atd11, some cells were harvested for TEM imaging.

In the vehicle control culture, barely any CD34⁺ cells coulddifferentiate into Mks by d8 (FIG. 9A (i), 9B). However, we observeddramatic induction of Mk differentiation (as assessed by CD41 expressionand polyploidization) in the d8 culture of CD34⁺ cells cocultured sinced0 with MkMPs (FIG. 9A (i), 9B). In addition, MkMPs promoted cellproliferation: the total cell number was increased 4.2 fold compared tovehicle control (FIG. 9B). We also examined if MkMPs could stimulatepartially differentiated HPCs. CD34⁺ cells were cultured in medium withor without TPO for 3 days and were then cocultured with MkMPs withoutTPO for 5 more days to d8. D3 HPCs from culture without TPO gave rise tovery few Mks in vehicle control culture, but MkMPs induced dramaticallyhigher (by >10,000 fold) differentiation into Mks (FIG. 9A(ii), 9D) witha concomitant 1.7-fold increased cell expansion (FIG. 9F) compared tovehicle control. In vehicle control cultures, d3 HPCs from culture withTPO developed into Mks by d8 even without further TPO stimulation.However, coculture with MkMPs resulted in 5.9-, 2.7- and 3.0-fold highernumbers of Mks with 2N, 4N and >8N ploidy (FIG. 9A(ii), 9E), althoughthe total cell number was not increased (FIG. 9G). Taken together, thesedata show that d12 MkMPs promote Mk differentiation of HSPCs atdifferent differentiation stages and that the effect is more pronouncedon more primitive, uncultured CD34⁺ cells.

In order to further characterize the Mks generated from CD34⁺ cellscocultured with MkMPs, the coculture was prolonged to d11. At d9, wefound that some Mks started to form proplatelets (FIG. 9C). At d11,cells were stained for β1 tubulin, vWF and serotonin to examineproplatelet structures, and the formation of α- and dense-granules,respectively. Fluorescent imaging demonstrated that Mks generated fromthe cocultures displayed normal microtubule proplatelet structures andsynthesized both types of platelet granules (FIG. 10A). TEM imaging(FIG. 10B) of Mks from d11 of coculture confirmed that numerous plateletgranules were packed in Mk cells, which also displayed thecharacteristic invaginated membrane system. These data demonstrate thatMks generated from coculture of CD34⁺ cells with MkMPs display normaldevelopmental characteristics.

Since d12 MkMPs contained ca.16% CD62P⁺ MPs with PMP characteristics, weexamined if PMPs generated from activated human platelets by thrombin orthe calcium ionophore A23187 could have an effect similar to that ofMkMPs. Compared to vehicle control, coculture of d3 HPCs (from cultureswith or without TPO) with either type of PMPs did not affect the Mkdifferentiation of HPCs compared to control (FIG. 9A(ii), 9D, 9E, 9F,9G). These data demonstrate that PMPs cannot promote Mk differentiationof HPCs, thus suggesting that the MkMP effect derives from the ˜84% ofCD62P⁻ MkMPs.

Although the protocol for generating MkMPs employs rigorous centrifugalenrichment and triple washing in IMDM medium, we wanted to verify thatthe impact of MkMPs in promoting the Mk differentiation of HSPCs was notdue to TPO attached to MkMPs. To this effect, we used a TPO ELISA assayto measure the amount of TPO carried by MkMPs and PMPs. We found thatthe total TPO carried into the cocultures by the MPs would result in5.2, 14, 27 pg/mL TPO in HSPC cocultures with PMPs (A23187), PMPs(thrombin) and MkMPs, respectively. This assumes that all TPO becomesavailable to all HSPCs, which is not true as we found that many MkMPsremain in culture for many days without being attached to cells. Nostudy has tested the effect of TPO at such low concentrations. Thelowest TPO level examined is 100 pg/ml, which has only a small impact onMk differentiation compared to saturation TPO levels. In support of theargument that the impact of MkMPs does not derive from the small amountof TPO carried into the coculture, we note that the TPO carried by thethrombin-generated PMPs (which had no Mk-differentiation impact on HPCs)was about half that carried by MkMPs.

This is the first study ever to show that true MkMPs have a biologicalrole in inducing megakaryocytic differentiation of HSPCs in the absenceof TPO and to do so in a physiological significant way leading to theformation of biologically active proplatelets as shown in FIG. 10. Thesedata support our claims for producing and using MkMPs for cells forapplications in Transfusion Medicine to promote in vivo and in vitro (exvivo) de novo megakaryopoiesis and platelet biogenesis without using TPOin in vivo applications.

Example 7. MkMPs are Produced Largely by Mature Mk Cells

We have shown above in Example 6 that Mks can shed CD41⁺CD62P⁻ MPs(i.e., MkMPs), and that exposure to fluid shear stress could enhancethis process by more than 20 fold in terms of numbers of MkMPs produced.In order to investigate MkMP generation in details under staticconditions, CD34⁺ HSCs were induced to differentiate into Mks aspreviously described. At d7 of cell culture, Mks were enriched(CD41⁺: >95%) and seeded in fresh medium at concentration of 200,000cells/mL. Concentration and CD62P expression of CD41⁺MPs in Mk cellculture were measured by flow cytometry from d8 to d12. The data showthat more than 85% of CD41⁺MPs in cell culture from d8 to d12 wereCD62P⁻, indicating that most of CD41⁺MPs were derived from Mks ratherthan platelet-like particles (PLPs) (FIG. 11A). The data also show thatMks at immature stage released MkMPs slowly from d8 to d11 and theconcentration of MkMPs in cell culture was increased by ˜50% every day(FIG. 11A). However, Mks shed more MPs and MkMP concentration wasincreased dramatically by 4.4 fold from d11 to d12 (FIG. 11A) whenmature Mks displayed extensive proplatelets but few PLPs were generated.

Example 8. Characterization of MkMPs Produced from Mature Mk Cells

We used MkMPs from d12 cell culture in the following studies. Throughsuccessive centrifugation, MkMPs were isolated from cell culture andprocessed for flow cytometric and electron microscopic analyses toobtain the size distribution of MkMPs. The flow cytometry datademonstrate that most of MkMPs were smaller than microbeads withdiameter of 0.88 μm (FIG. 11B). This result was confirmed by TEM and SEManalyses (FIGS. 11C and 11D). In addition, TEM micrograph also showsthat very few particles were smaller than 100 nm, indicating that we didnot enrich exosomes (40-100 nm) with MkMPs from cell culture using ourcentrifugation protocol. We observed that TEM staining of some MkMPs(indicated by black arrow) was very light while the staining of others(indicated by arrow head) was very dense (FIG. 11C). In addition, someMkMPs (indicated by white arrow) even carried several smaller particlesinside them (FIG. 11C). These observations indicate that MkMPs were notuniform with respect to their size and internal content. As shown by SEMmicrographs, MkMPs were not spherical and their membrane was not smooth(FIG. 11D).

Example 9. MkMPs Target with Even Higher Effectiveness the MostPrimitive Hematopoietic Stem Cells, Namely the CD34⁺ Lin⁻ Cells

Significantly, MkMPs also promote the expansion of the CD34⁺ HSPCs. Inexample 6, we showed that MkMPs could induce and enhance differentiationof CD34⁺ HSPCs and partially differentiated HPCs from d3 and d5 Mkculture to Mks that were functional to project PPT and synthesize bothα- and dense-granules without additional exogenous TPO stimulation.Here, we wanted to show that MkMP cells target also the leastdifferentiated of the HSPC CD34⁺ cells, the Lineage negative (Lin) cellby removing the committed Lineage positive (Lin⁺) cells namely the CD2⁺,CD3⁺, CD11b⁺, CD14⁺, CD15⁺, CD16⁺, CD19⁺, CD56⁺, CD123⁺, or CD235a⁺cells. This was achieved using the lineage cell depletion kit fromMiltenyi Biotec. A total of 60,000 CD34⁺ Lin⁻ cells were incubated with10 MkMPs/cell in 50 μL IMDM medium for 1 hour at 37° C. to enhance thecontact between MkMPs and target cells. After that, coculture of MkMPswith CD34⁺ Lin⁻ cells were diluted in 600 μL IMDM medium supplementedwith 5% BIT9500 and 50 ng/mL rhSCF, and cultured at 37° C. and 20% O₂.All cocultures were maintained for 8 days before harvest for ploidyassay and analysis by flow cytometry. CD34⁺ Lin⁻ cells at d0 and cellsfrom coculture at d3, d5 and d8 were stained with CD41, CD34, CD11b andCD235a antibodies and analyzed by flow cytometry. Flow cytometryanalysis shows that MkMP coculture had a large amount of Mks with 2N, 4Nand >=8N ploidy classes while very few Mks were found in the vehiclecontrol culture (FIGS. 12A and 12B). In addition, there were more totalcells in MkMP coculture than in control culture (FIG. 12B). Theseresults demonstrate that MkMPs were able to induce differentiation ofLin⁻CD34⁺ cells into the Mk lineage.

In order to find out what are the CD41⁻ cells are in the MkMP coculture,cells from the cocultures and control cultures were stained withanti-CD34, CD41, CD11b and CD235a antibodies to identify HSPCs, Mks,granulocytes and erythrocytes, respectively, and analyzed by flowcytometry. The results show that very few cells differentiated intogranulocytes or erythrocytes in either the vehicle control culture orthe MkMP coculture, indicating that MkMPs could not induce HSCsdifferentiation to these two lineages. This shows that the effect ofMkMPs on HSPCs is specific to the Mk lineage differentiation andsupports the claims related to outcome specificity for in vivoapplications, i.e., that MkMPs promote ONLY the Mk differentiation ofHSPCs.

In the MkMP coculture, the percentage of CD41⁺ cells increased from 0%at d0 to ˜47% at d5 and plateaued after d5, indicating that 5 days aresufficient for HSCs to commit to Mk lineage induced by MkMPs. From theploidy analysis, the percentage of CD41⁺ cells in coculture at d8 wasabout 19% which is lower than the ˜48% obtained from the surface markerstaining analysis. This could be due to the assay methodology since wealways obtained lower CD41⁺ percentages from ploidy analysis thansurface marker staining. This could be also contributed partially by thepossibility that CD41⁻ cells with CD41⁺ MkMPs attached were detected byflow cytometry as CD41⁺ cells in surface-marker staining analysis butnot in ploidy assay. Compared to the vehicle control culture, thecoculture also had a higher percentage of CD34⁺ cells at d8 (47% vs.28%) and based on the total cell number obtained from ploidy assay,there were more CD34⁺ cells in coculture than control culture (108 k vs.27 k, FIG. 12B). These results show that MkMPs have two biologicaleffects on HSPCs: promote expansion of CD34⁺ cells and induce Mkdifferentiation of CD34⁺ HSPCs. These findings are novel, and supportthe use of MkMPs for cell for applications in Transfusion Medicine andstem-cell transplantation.

Example 10. Target Specificity: MkMPs could not Trans-DifferentiateHuman Granulocytes, MSCs and HUVECs into Mk Cells

Previous studies have demonstrated that certain types of cells cantrans-differentiate into other unrelated types of cells It is possiblethat cell-derived microparticles mediate trans-differentiation. Forexample, microparticles derived from lung endothelial cells inducedtrans-differentiation of bone marrow cells into endothelial cells. Toinvestigate if MkMPs could trans-differentiate other cell types intoMks, we tested human granulocytes, MSCs and HUVECs, all of which areencountered by MkMPs in the bone marrow environment or in circulation.

A total of 60,000 HUVECs (human umbilical cord vascular endothelialcells; passage 3-5, obtained from ATCC), MSCs (mesenchymal stem cells;passage 2-4, donation from Prof. Xinqiao Jia, Univ of Delaware) orenriched CD15⁺ granulocytes were incubated with 10 MkMPs/cell in 50 μLIMDM medium for 1 hour at 37° C. to enhance the contact between MPs andcells. After that, coculture of MkMPs with MSCs and granulocytes werediluted in 600 μL IMDM medium supplemented with 5% BIT9500 and 50 ng/mLrhSCF, and cultured at 37° C. and 20% O₂ and coculture of MkMPs withHUVECs were diluted in 600 μL growth medium without any endothelial cellgrowth factors. All cocultures were maintained for 8 days before harvestfor ploidy assay and analysis by flow cytometry. Flow cytometry analysis(FIG. 13) shows that no CD41⁺ or polyploid cells were observed after 8days of coculture of MkMPs with granulocytes, MSCs or HUVECs, indicatingthat MkMPs could not trans-differentiate these types of cells into theMk lineage and thus the action of MkMPs is target-specific. MkMPs mayonly affect the fate of HSPCs but not MSCs or other mature cells likegranulocytes or HUVECs. This target specificity may be caused by theinability of these three types of cells to internalize MkMPs becausethey are lacking suitable surface receptors to mediate MP uptake or theinability of signaling molecules carried by MkMPs to inducetrans-differentiation. These novel findings support our claim for theexquisite specificity of MkMP to ONLY target HSPCs and no other relatedcells like HUVECs, MSCs, or granulocytes. This shows that MkMPs can beused for in vivo animal or human transplantation to specifically anduniquely target HSPCs in vivo, and thus support our claims to thateffect.

Example 11. MkMPs Promote Mk Differentiation Through Transfer of the RNACarried by the MkMPs

Several studies have reported that signaling molecules carried by MPs,including ESC-derived MPs, MSC-derived MP and PMPs, are RNA (mRNA and/ormiRNA) and MPs exert their biological function through RNA transfer totarget cells. To investigate if MkMPs induce Mk differentiation of HSPCsthrough RNAs, MkMPs were treated with RNase to degrade if possible theRNA carried by these MkMPs and cocultured with HSPCs, similar to whatprevious studies have reported. Two different commercial RNases, theRNase A/T1 cocktail from Ambion and the RNase ONE™ from Promega, wereused in this study and ploidy analysis was used to examine the effect ofRNase treatment. As expected, we found that MkMPs without RNasetreatment induced Mk differentiation of HSCs and increased the totalcell number while no differentiation was observed in vehicle controlculture (FIG. 14). The numbers of 2N, 4N and >=8N Mks as well as totalMks and other cells was decreased by ˜50% by either type of RNasetreatment (FIG. 14). RNase treatment did not totally abolish the effectof MkMPs on HSCs. This could be due to incomplete digestion of RNA,especially miRNA which is more RNase-resistant than mRNA and has beenproposed to be mainly responsible for the biological effects of otherMPs. Another possible reason is that proteins carried by MkMPs may bealso involved in inducing Mk differentiation, like ESC MPs and tumor MPswhose biological functions are dependent on both of protein and RNAtransfer. Here, we demonstrated that MkMPs induce differentiation ofHSPCs towards to Mks, partially at least, through the carried RNAs.

Example 12. MkMPs Interact with Target Cells Through Endocytosis andMembrane Fusion

Next, we examined detailed mechanisms through which MkMPs exerted theirimpact on HSPCs. Although it has been proposed that MP may interact withor be taken up by target cells through direct fusion and endocytosis,there is no information disclosed as to how MkMPs interact and maytransfer molecules they contain to target cells. To investigate themechanisms by which MkMPs interact with target cells and notably HSPCs,MkMPs were stained with cell cytoplasmic tracker dye CFDA-SE and thencocultured with hematopoietic progenitor cells (HPCs) from d3 of Mkculture. After cultured for certain time as indicated below, cells wereprocessed for flow cytometric and microscopic analyses.

The coculture was firstly analyzed by flow cytometry to examine thekinetics of MkMP binding to cells. At each indicated time point, somecells were harvested from coculture with CFDA-SE stained MkMPs formeasurement of mean fluorescence intensity (MFI) of CFDA-SE. The resultsshow that CFDA-SE MFI of cells increased dramatically within one hour ofcoculture and reached the maximum level at one hour (FIG. 15). Ascoculture continued, CFDA-SE MFI of cells decreased and plateaued around24 hours (FIG. 15). These data indicate that MkMPs bind to cell surfacevery quickly within one hour. After one hour of coculture time, CFDA-SEMFI of cells decreased and this could be caused by the dilution duringcell proliferation and the possibility that some MkMPs may dissociatefrom the cell surface.

Since flow cytometry cannot differentiate MP binding to cells and uptakeof MkMPs by cells, coculture of MkMPs and HPCs was examined underconfocal microscopy to directly visualize the interaction between livecells and MkMPs. After 3-5 hours of coculture, we observed that mostHPCs contained CFDA-SE dye of variable intensity (FIGS. 16B and 16C) andthis indicates uptake of MkMPs and thus transfer of dye from MkMPs tocells. Further analyses demonstrated that uptake of MkMPs was throughtwo different mechanisms: endocytosis and direct fusion. We found thatsome cells contained various numbers of distinct CFDA-SE fluorescencedots representing intact MkMPs that were not clearly associated with thetarget-cell membrane (FIGS. 16B and 16C, arrow head and FIG. 17),suggesting that these MkMPs were inside the cells. To confirm thisfinding, images of cells from different confocal planes with 0.4 μminterval were collected to make up a z-stack and 3D images werereconstructed from z-stacks. As demonstrated in 3D images of cells fromcoculture, intact MkMPs (concentrated green dots) “move” at a shorterdistance than the cell edge, delineated by a dim green signal, when thecell rotates along a central axis, indicating that these MkMPs wereinside the cell rather than on the cell membrane. These data demonstratethat intact MkMPs were internalized by cells through cell endocytosis

In addition to cell internalizing intact MkMPs, we also observed that agradient of CFDA-SE dye distributed inside some cells and this gradientstarted from one MkMP on the cell membrane as indicated by the highlyconcentrated dye (FIG. 17). This CFDA-SE dye gradient may have resultedfrom MkMPs fusing with the cell membrane, and following that, CFDA-SEdye being discharged directly from the MkMP into the cell cytoplasm. Tofurther confirm that direct fusion mediates this dye gradient formation,cells from MkMP coculture was examined using scanning electronmicroscopy (SEM) to capture the interaction between MkMPs and cells indetails.

SEM micrographs of cells from coculture (3-5 hours) show that MkMPsinteracted with through a membrane fusion process (FIG. 18A). Some MkMPswere bound to the cell surface and some were partially fused into thecell (FIG. 18A), confirming that direct fusion took place in MkMPcoculture. SEM micrographs reveal 4 gradual stages (FIG. 18A) throughwhich MkMPs fused into cells based on the relative volume of MkMPs leftoutside of the cell membrane. At the 1^(st) stage, MkMPs attached to thecell surface by remaining as an intact sphere; at the 2^(nd) stage,MkMPs were partially incorporated and less than half of their body wasmerged with cells; at the 3^(rd) stage, MkMPs were half fused into HPCswith formation of lamellipodia-like structures extending from the MkMPat the contact area with the cells (FIG. 18A, white arrow); at the4^(th) stage, whole MkMPs were fused into cells and an almost flat MkMPmembrane with wrinkles was left on the cell membrane. To the best of ourknowledge, these four stages of MP-cell fusion and lamellipodia-likestructures have not been reported in the literature before. We alsoquantified the percentage of MkMP-cell interaction at each stage after 3and 5 hours of coculture (n=2 biological replicates). Most (˜63%) offusion events were at the 1^(st) stage and very few (˜9.7 and ˜2.8%respectively) were at the 3^(th) and the 4^(th) stages when MkMPs werecocultured with HPCs for 3 hours (FIG. 18B). The fusion event at the1^(st) stage decreased to ˜41% while the percentage of fusion events atthe 3^(rd) and 4^(th) stages increased to ˜20% and ˜12.9%, respectively,after the coculture time increased to 5 hours (FIG. 18B), indicatingthat MP-cell fusion took place continuously in the MkMP coculture.

In order to capture the internal structures of the MkMP-cell fusion,cells from coculture were processed for TEM analysis. We captured MkMPsbound to cell surface in TEM micrographs (Data not shown). However, wedid not successfully capture MkMP-cell fusion events at all four stagesidentified in SEM micrographs. A possible reason could be that since TEMexamines one ultrathin slice of cell samples, the chance that theultrathin slice contains one MkMP is low. Nevertheless, two MkMPs fusedwith cells were found in TEM micrographs (FIG. 19). These particles wereconsidered as MkMPs rather than MPs from the cell themselves or part ofthe cell body based on that they had totally different internal textureand composition from the cell body. The MkMP in the FIG. 19A showslamellipodia-like structures (white arrow), which we found previously inSEM micrographs on the cell membrane around the contact area with theMkMP. We found that two MkMPs interacted with one cell in the second TEMmicrograph (FIG. 19B). The top MkMP is only binding to cell surfacewithout any fusion sign (FIG. 19B). In contract, the membrane (whitearrow) between the MkMP at the bottom and the cell body was diminished,demonstrating that the MkMP was partially fused into the cell (FIG.19B).

This is the first disclosure ever as to of the mechanism by which MkMPsinteract with and target HSPCs and supports our claims for the use ofunmodified and modified MkMPs.

Example 13. Generation of MkMPs (Termed CMPs) from the HumanMegakaryocytic Cell Line CHRF and Demonstration that CMPs can AlsoInduce and Promote Mk Differentiation of HSPCs

CMPs were generated from d3 phorbol 12-myristate 13-acetate(PMA)-induced CHRF cells. 40000 HSCs were coculture with or without CMPsat the concentration of 50 MPs/cell for 1 hr at 37° C. in 50 mL IMDM inthe medium to increase CMP-cell contact. Cells with or without CMPs werethen diluted into 600 mL medium without thrombopoietin (IMDM, 5%BIT9500, 50 ng/mL rhSCF) at 37° C. and 20% O₂ for 8 days. Cell wereharvested on d8 for CD41 and DNA staining and analyzed of CD41expression and ploidy by flow-cytometry (FACSAria II, BD bioscience).Collected CMPs were cocultured with HSCs at the concentration of 50MPs/cell for 8 days. From the analysis of ploidy flow-cytometry, FIG.20A shows that HSCs in coculture became Mks with the properties of CD41expression and polyploidy. The number of Mk of coculture is larger thanMk of control (FIG. 20B). These data show that CMPs are able to induceMk differentiation of HSCs. Although CMPs may not be suitable forroutine applications in human therapy, they can be used to test variousprocesses necessary for the application of primary MkMPs, as asurrogate.

Example 14. RNase Treatment is Effective in Eliminating (“Unloading”)the Native RNA in Megakaryocytic Microparticles so that they can beLoaded with Desirable Molecules for Delivery to Target HSPCs

We used CMPs as a model for primate MkMPs. In example 11, we have shownthat RNase treatment can partially abrogate the impact of MkMPs onHSPCS. Here we optimized the process of RNase treatment to remove theRNA content of CMPs. HSPCs were coculture with CMPs, or RNase-treatedCMPs, or without CMPs (Control) for 8 days. In detail, CMPs werecollected as mentioned previously, and were treated with 1 U/mL RNaseA/T1 (Ambion) or 10 U/mL RNase ONE (Promega) under the condition of 37°C. for 1 hr. After that, 10 U/mL RNase inhibitor, SUPERase-In (Ambion)were added to prevent further reaction from RNase. CMPs were then washedwith IMDM and collected by ultracentrifugation at 25000 rpm, 4° C. 60000of HSCs were cocultured with CMPs, or RNase-treated CMPs, or withoutCMPs at the concentration of 50 MPs/cell for 8 days in the IMDM mediumsupplemented with 5% BIT9500, 50 ng/mL rhSCF, but withoutthrombopoietin. Cell were harvest at d8 for CD41 and DNA staining.Analysis of cell ploidy and CD41 expression and Mk cell number wereperformed by flow-cytometry (FACSAria II, BD bioscience). HSPCscocultured with CMPs without RNase treatment became Mks with polyploidyand CD41 positive and the number of Mks with CMPs coculture is higherthan Mks of vehicle control. However, here, the number of Mks of bothRNase-treated CMPs cocultures decrease, compared to the Mks in CMPcoculture (FIG. 21). These data show that the treatment of RNase A/T1 orONE on CMPs decreases the CMPs ability to induce Mk differentiation ofHSCs. Since the function of RNase is to degrade RNA from CMPs, thesedata show that an optimized RNase treatment can be used to effectively“unload” the native RNA from the MkMPs.

Example 15. Loading of pmaxGFP DNA into CMP or MkMP by Electroporation

To load exogenous material into MP, we choose plasmid DNA as modelmolecule for loading into MkMPs. There is no prior art on the loading ofany microparticles (MkMPs or any other MPs) with exogenous molecules,and thus the following enabling data support our claims for themodification and loading of MkMPs and all MPs with exogenous moleculeslike DNA, RNA, proteins, non-protein morphogens and drugs. Such loadingprocess could not be anticipated by someone skilled in the art since MPsare very different entities from cells.

We used a commercially available DNA plasmid (PmaxGFP DNA; Lonza), whichwe labeled with red fluorescent dye Cy5 using the Label IT® Nucleic AcidLabeling Kit (Minis) based on the manufacturer's protocol. 2 μg ofCy5-pmaxGFP were electroporated into 10⁶ CMPs or MkMPs by using AMAXANucleofector™ II Device (Lonza) with program T03 or U08, respectively.The procedure of electroporation was followed by manufactural protocol.CMPs or MkMPs were then incubated at 37° C. for 30 min. After 3 times ofwash of CMPs or MkMPs with IMDM medium by ultracentrifugation at 25000rpm, 4° C. for 1 hr each, the loading efficiency of Cy5-pmaxGFP intoCMPs or MkMPs were analyzed with flow-cytometry FACSAria II based on Cy5fluorescence. Electroporation of fluorescent-labeled plasmid DNA(Cy5-pmaxGFP) into MkMPs and CMPs have been performed on AMAXANucleofector™ II Device using the specific program U08 and T03,respectively. Base on Cy5 signal, flow-cytometry analysis shows that95.9% of MkMPs and 97.1% of CMPs were Cy5 positive (FIG. 22), whichindicates that both MkMPs and CMPs were loaded with pmaxGFP DNA byelectroporation. A summary of the procedure for loading MkMPs and otherMPs with exogenous molecules is depicted in FIG. 23. Our datademonstrate that MkMPs and other MPs can be loaded with exogenousmolecules and thus methods other than electroporation can be used forcarrying out this task. Such methods would include but are not limitedto lipofection, virus-mediated transfer, receptor mediated transfer,synthetic particle mediated transfer, and direct injection.

Discussion of our Disclosed Data for the Support of our Claims

We show for the first time that shear stress dramatically increased MkMPgeneration by 30-40 fold. Flaumenhaft et al. demonstrated that the CD41⁺MPs in human plasma are mainly derived from Mks rather than activatedplatelets [5]. When mature Mks enter BM sinusoids and are exposed toshear circulatory forces, numerous MkMPs are likely formed. PMPgeneration from platelets on immobilized vWF surfaces is also promotedby high shear. While the cellular mechanisms leading to membranevesiculation and MP release remain an active research field, studiesfrom PMP biogenesis suggest that PS externalization and caspase-3activation play an important role in MP generation. In our study, wefound that caspase-3 activation and PS externalization were enhanced byshear stress, thus suggesting that shear-stress enhanced MkMP generationmay be mediated by PS externalization and caspase-3 activation. Thelatter is supported by the data from the caspase-3 inhibition assays.

The physiological function for MkMPs was also investigated. Wedemonstrated that MkMPs promote the survival and Mk differentiation ofHSPCs in the absence of added TPO. Thus, one possible role for MkMPs incirculation may be to promote differentiation of circulatory HSPCs orperhaps re-enter the hematopoietic BM compartment aiming to target HSPCsfor accelerated megakaryopoiesis under stress. Biological roles havebeen reported previously for other MPs, but never before for MkMPs. Forexample, MPs generated during macrophage differentiation of THP-1 cellsinduced differentiation of resting THP-1 cells into macrophages throughmiRNA-223 transfer [2].

MPs may serve in several different roles in biological processes sincebioactive molecules carried by MPs are concentrated and can travel longdistances with protection from degradation. Here, we demonstrated thatMPs generated by mature Mks induce Mk differentiation of very primitiveHSPCs (CD34⁺ Lineage⁻ cells). In addition, we also investigated if MkMPscould transdifferentiate other types of cells, including MSCs, HUVECsand granulocytes and found out they could not. All of these three typesof cells are likely to be in contact with MkMPs in vivo.

We also demonstrated mechanisms through which MkMPs exert theirbiological effect. Two main questions were examined here: how do MkMPsinteract with target cells, and what might be the signaling moleculescarried by MkMPs. Three different mechanisms have been reported in theliterature to explain how cell-derived MPs and target cells interact.The interaction always starts from MP binding to cells, which requiresrecognition between receptors and ligands on the membrane surface of MPsand cells. This ligand-receptor recognition is the major reason for thetarget specificity of MPs. MP binding could be unstable, leading todissociation of MPs from cell surface, or stable, ending in uptake ofMPs by cells. The signaling from temporary or persisting binding of MPscould be enough to regulate cell fate. For example, transfer of CCL5from PMPs to activated endothelial cells only happens during transientinteraction rather than firm attachment between PMPs and endothelialcells under flow condition [6]. It is possible that this temporarybinding of MkMPs may have an effect on target HSCs. In addition, we haveshown that some MkMPs were taken up by cells through direct fusionand/or endocytosis. After we treated MkMPs with two different RNases todigest the RNA carried by MkMPs, the numbers of Mks with differentploidy levels decreased by half in the coculture with treated MkMPscompared to MkMPs without treatment. This result demonstrates thathorizontal transfer of RNA is crucial for the observed biological effectof MkMPs. Transfer of RNA requires uptake of MkMPs by cells followingstable binding and thus the RNase treatment results provide additionalevidence that MkMPs are taken up by cells. Through confocal microscopy,SEM and TEM analyses of MkMP coculture, we demonstrated that both directfusion and endocytosis were involved in uptake of MkMPs. Moreover, forthe first time, using SEM analysis we dissected the MP fusion process asproceeding through 4 distinct stages. The lamellipodia-like structuresduring MP fusion were observed and reported for the first time. Theseresults contribute to our limited understanding of MP uptake by cells,which is important regarding the delivery of biological molecules (suchas but not limited to RNA, DNA, proteins, lipids) as well other organicmolecules and drugs by modified MkMPs for delivery to stem cells. Avariety of cellular contents may be delivered to target cells includingRNA, DNA, proteins, lipids, phospholipids, non-protein morphogens,non-biological materials, organic molecules, non-organic molecules,synthetic drugs or natural drugs.

It has been reported that MPs have several biological functions and playan essential role in various physiological and pathophysiologicalprocesses. Different MPs may have a role in blood coagulation,inflammation, angiogenesis, tumorigenesis, cell differentiation andmaturation. Ratajczak et al. showed that MPs from embryonic stem cells(ESCs) when cocultured with hematopoietic progenitor cells (HPCs) resultin upregulated expression of early marker of pluripotent (Oct-4, Nanogand Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2)markers [7]. Their data suggest that the effect is mediated by RNAs andproteins in the MPs. In another example, MPs from stimulated orapoptotic T lymphocytes harbored sonic hedgehog (Hh) morphogens and wereable to induce K562 cells (a cancer cell line) differentiation towardsthe Mk lineage and promote Mk differentiation of CD34⁺ HSPCs whencultured in the presence of thrombopoietin [8]. Hh was necessary forthese effects. Our invention is very distinct and could not have beenanticipated by these findings for several reasons explained in detailbelow. First, we employ MkMPs and not T-cell derived MPs. Second, ourMkMPs do not require Hh morphogens for the effects on HSPCs. Thirdly,this report requires that CD34+ cells are cultured in the presence ofTPO, while our invention does not. Fourth, in this report, they showthat Hh containing T-cell MPs promote Mk differentiation but not theproduction of proplatelets or platelets or platelet-like particles.

Mk cells are great sources for MPs since they have much larger cellvolume and massive membranes compared to other types of cells. In thepresent invention, MkMPs are first unloaded from the endogenous RNAs andthen reloaded with the desirable RNAs, DNAs, proteins or other moleculesfor delivery to the target HSPCs. The present invention engineerscell-derived MkMPs where endogenous RNAs are removed from MPs usingRNase treatment and exogenous molecules (plasmid DNA here) are loadedinto MPs directly using electroporation. This process can be applied toother MPs beyond MkMPs and thus, we disclose for the first time a uniqueand powerful method for unloading the natural RNA cargo of MPs in orderto re-load them with desirable “cargo”, i.e., any desirable moleculesfor delivery to target cells including HSPCs.

In addition to view cell-derived MPs as a tool or a vesicle to delivertherapeutic drugs, MPs from specific cells (like endothelial cells,mesenchymal stem cells or other types) have unique biological functionon target cells and other inventors used these MPs as a type of drugs totreat certain types of diseases. In U.S. patent application US20120321723 A1, the inventors found MPs from stem cells, preferably abone marrow-mesenchymal stem cell, a glomerular mesenchymal stem cell ora non-oval liver stem cell, exert anti-tumor effect when administered toa tumor patient.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Source of Hematopoietic Stem and Progenitor Cells (HSPCs) andGeneration of Megakaryocytes (FIG. 24).

Multiple sources of HSPCs, including but not limited to autologous orallogeneic CD34+ cells from bone marrow, peripheral blood or cord blood,are cultured and differentiated to megakaryocytes using a publishedprotocol such as the one published in [9], but many other protocols andtheir variations can be used. Several such protocols have been disclosedin the scientific and patent literature. HSPCs can be also obtained fromembryonic cells or from induced pluripotent stem (iPS) cells.

2. Generation of MkMPs, PLPs or PPTs from Mature or MaturingMegakaryocytes (FIG. 24).

To generate MkMPs or MPs from other cells, shear or other biomechanicalforces are applied to the megakaryocytes to generateproplatelets/preplatelets (PPTs), platelet-like particles (PLPs) andmegakaryocytic microparticles (MkMPs). The shear flow can be laminar orturbulent and flow application can be carried out in a parallel channelbioreactors, or mixed bioreactor where the cells are either freelysuspended or growing on microcarriers (small beads of 100-1000 micronsin diameter) where the cells can attach to for growth and maintenance.The intensity of biomechanical forces can be controlled by controllingthe flow rate or the agitation/mixing rate as is known to someoneskilled in the art, such as in described in Refs. [9-13]. The use ofagitated or mixed bioreactors of freely suspended cells or cellsattached to microcarriers for producing MPs is disclosed for the firsttime here. Microccariers are small particles, mostly spherical but notonly, whose surface is appropriately modified for allowing cells toattach and grow on the particle surface. [14, 15] Some microcarriers arealso porous allowing cells to grow into the microcarriers. Both types ofmicrocarriers are used for growing cells that require or prefer surfaceattachment and where the culture can be done both under staticconditions but more typically in stirred bioreactors or vessels thatenable scale up and quality control by controlling the cultureconditions using various sensors such as for pH, dissolved oxygen,nutrient concentrations and metabolite concentrations. [12, 14-16]Tubular or channel bioreactors or microreactors or microfluidic reactorscan be used to culture all types of cells and also to expose varioushuman or mammalian cells to biomechanical forces. [13, 16-20] The use ofsuch bioreactors for generating MPs or MVs or any other particle frommammalian or other cells is disclosed for the first time here. The useof such bioreactor systems for generating MPs or MVs or any otherparticle from mammalian or other cells could not have been anticipatedby someone skilled in the art.

3. Separation, Purification and Storage of MkMPs, PLPs, and PPTs butAlso MPs from Other Cell Types (FIG. 24).

Three types of particles, PPTs, PLPs and MkMPs, are collected andenriched from static cell culture and flow application using successivedifferential centrifugations as described in Ref. [9], but several othercentrifugation, ultracentrifugation, elutriation, sedimentation and/ormembrane protocols can be used as is well known by those skilled in theart [21-24]. MkMPs can be frozen as is known in the art of freezinghematopoietic stem cells for transplantation therapies. [25-27] PLPs orPPTs can either be stored at room temperature or used immediately. PLPsor PPTs can be also frozen using methods as for freezing MkMPs.

4. Modification of MkMPs for the Delivery of “Cargo” Molecules (DNA,RNA, Proteins, Etc) to HSPCs Both In Vivo and Ex Vivo (FIG. 24).

To engineer MkMPs, RNase is used to remove endogenous RNAs inside themicroparticles by incubation of RNase and microparticles at 37° C. Afterthat, the desirable RNAs, DNAs, proteins or other therapeutic drugs areloaded into MkMPs using electroporation (e.g., using Nucleofectionperformed on AMAXA Nucleofector® II Device, Lonza) or lipofection orother methods used in the modification of whole cells, but here appliedto MkMPs.

5. Applications of the produced unmodified MkMPs, PLPs or PPTs (FIG.24):

a. Hematopoietic transplantation for the reconstitution of HSPCs in vivoby intravenous infusion or co-infusion with HSPCs, either autologous orallogeneic. This is to enhance the in vivo expansion of HSPCs (byMkMPs), but also the in vivo megakaryopoiesis and platelet biogenesis(by MkMPs, PLPs, and PPTs). The latter two processes are beneficial topatients undergoing chemotherapy or patients with genetic or idiopathicdisorders.b. Ex vivo production of proplatelets or platelets. MkMPs also can beused as differentiation inducing reagent in ex vivo megakaryocyticdifferentiation and platelet production. The collected PPTs and PLPs canbe used in clinical transfusion and intravenously infused to patientswho need platelets, including those suffering severe thrombocytopeniadisease, idiopathic or due to chemotherapy.6. Applications of Modified MkMPs (FIG. 24).

The engineered MkMPs can be used in two different ways to deliverdesirable materials (DNA, RNA, proteins, morphogens, or drugs) intotarget cells, including the HSPCs. One way is to infuse these engineeredmicroparticles directly into the patient circulation. The other way isto coculture these microparticles with CD34+ HSPCs or other target cellsex vivo for 2-5 days and then transfuse those cells from coculture intopatients.

Although preferred embodiments of the disclosure are illustrated anddescribed in connection with particular features, it will be apparent tothose skilled in the art, that the invention can be adapted for use fora wide variety of applications. Various features of the disclosure havebeen particularly shown and described in connection with illustratedembodiments. However, it must be understood that the particularembodiments merely illustrate and that the invention is to be given itsfullest interpretation within the terms of the claims.

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The invention claimed is:
 1. An in vitro method to generate particlesfor platelet function comprising the steps of: a. generating culturedcells selected from the group consisting of megakaryocytes and immaturemegakaryocyte cells; b. exposing said cultured cells to a biomechanicalstress that generates at least one particle for platelet function; c.collecting said particles from a supernatant of the cultured cells instep b; d. loading said particles obtained from step c with nativecellular content(s) of said cultured cells; e. providing said loadedparticles to a co-culture with at least one target cell to provide atransfer of said cellular content(s) to said target cells, wherein saidtarget cell is selected from the group consisting of hematopoietic stemcells, and progenitor cells; wherein said co-culture with said targetcells is performed in the presence of a culture medium lackingthrombopoietin; and wherein said transfer enables effectivedifferentiation of said target cells and production of additionalparticles for platelet function selected from the group consisting ofmegakaryocyte microparticles, proplatelets, preplatelets, platelet-likeparticles and megakaryocyte extracellular vesicles.
 2. The method ofclaim 1, further comprising loading said particles in step d withexogenous cellular content(s) selected from the group consisting of RNA,DNA, proteins, lipids, phospholipids, non-protein morphogens,non-biological materials, organic molecules, non-organic molecules, andsynthetic or natural drugs.
 3. The method of claim 2, further comprisingloading said exogenous cellular contents by a transfection method. 4.The method of claim 3, wherein said transfection method is selected fromthe group consisting of lipofection, nucleofection and electroporation.5. The methods of claim 1, wherein said particles for platelet functionproduced are stored frozen.
 6. The method of claim 1, further comprisingunloading the native RNA of said particles from step c using an RNasetreatment.
 7. The method of claim 1, further comprising unloading thenative DNA of said particles from step c using a DNase treatment.
 8. Themethod of claim 1, further comprising unloading the native protein ofsaid particles from step c using a protease treatment.
 9. The method ofclaim 1, further comprising unloading the native lipid of said particlesfrom step c using a lipase treatment.
 10. The method of claim 1, whereinsaid biomechanical stress is selected from the group consisting of shearstress, normal stress, laminar flow stress and turbulent flow stress,wherein exposure of said cultured cells to the biomechanical stressincreases DNA synthesis, cell ploidy and generation of said particlesfor platelet function selected from the group consisting ofmegakaryocytic extracellular vesicles, proplatelets, preplatelets,platelet-like particles and megakaryocyte microparticles.
 11. The methodof claim 2, wherein the loaded particles are stored frozen.
 12. Themethod of claim 6, further comprising loading the unloaded particleswith an exogenous RNA.
 13. The method of claim 7, further comprisingloading the unloaded particles with an exogenous DNA.
 14. The method ofclaim 8, further comprising loading the unloaded particles with anexogenous protein.
 15. The method of claim 9, further comprising loadingthe unloaded particles with an exogenous lipid.